Stem Rust Resistance Gene

ABSTRACT

The present invention relates to a transgenic plant which has integrated into its genome an exogenous polynucleotide encoding a polypeptide which confers resistance to one or more races of  Puccinia  gram in is r. sp. tTitici, such as the Ug99 group of races  Puccinia graminis  f. sp.  tritici.

FIELD OF THE INVENTION

The present invention relates to a transgenic plant which has integratedinto its genome an exogenous polynucleotide encoding a polypeptide whichconfers resistance to one or more races of Puccinia graminis, such asthe Ug99 group of races of Puccinia graminis f. sp. tritici.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED

A Sequence Listing is provided herewith as a Sequence Listing XML,“522819US01 Stem rust resistance gene” created on May 4, 2023 and havinga size of 100 KB. The contents of the Sequence Listing XML areincorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

The fungal pathogen Puccinia graminis Pers. f. sp. tritici (Pgt), thecausal agent of wheat stem rust, is a major threat to wheat production,in part because of the level of devastation it can inflict on wheatcrops with near complete losses in severe epidemics. Although rustresistant cultivars had controlled the disease for over 40 years, theappearance in Uganda in 1999 of Pgt race TTKSK, commonly known as Ug99,and the subsequent spread of its derivatives to other parts of Africaand the Middle East, has highlighted the continuing danger of stem rust.Ug99 and derivative strains are virulent on plants containing the stemrust resistance gene Sr31, which had previously provided effectiveresistance for over thirty years (Ellis et al., 2014), as well as manyother common stem rust resistance (Sr) genes (Singh et al., 2011). Therecent appearance of another Pgt race (different to the Ug99 lineage) inGermany and Ethiopia in 2013-2014 attacking important commercial wheatvarieties has raised further serious concerns for wheat production.While an international collaboration called the Borlaug Global RustInitiative (BGRI) has since been actively involved in strategies tocontrol stem rust, changes in global climate and warming can potentiallyopen additional areas for rust epidemics which were previouslyconsidered safe from stem rust (Chakraborty et al., 2011).

The control of wheat rust is dependent on the incorporation of effectiveresistance genes during breeding and combinations of multiple stem rustresistance genes are crucial for providing durable resistance, whichnecessitates the identification of new resistance genes. Wild andcultivated relatives of wheat provide an important pool of new geneseffective against wheat rust pathogens. Cereal rye (Secale cereale) isone such source and several rye genes have been used in breeding rustresistant bread wheat and triticale. Indeed, the Sr31 resistance genewas introgressed from rye as a full chromosome substitution for wheatchromosome 1B or as a translocation of the short arm of rye chromosome 1(1RS) from the rye cultivar Petkus (Zeller, 1973) to the long arm ofwheat chromosome 1B (1BL) and conferred stem rust resistance for over 30years in the field (Ellis et al., 2014). Sr50 (previously known as SrR)was also introgressed into wheat as translocations of 1RS to the longarms of wheat chromosomes 1B and 1D, but sourced from the rye cultivarImperial (Shepherd 1973; Mago et al., 2004). A third stem rustresistance gene, also known as Sr1RS^(Amigo), was introgressed as a 1RStranslocation from rye cultivar Insave to wheat chromosome 1A incultivar Amigo (Zeller and Fuchs, 1983). Because these rye genes gaveresistance to all known Pgt strains, it was not clear whether theyrepresented different resistance specificities. Both Sr31 and Sr50 areassociated with a cluster of genes on 1RS (Mago et al., 2004 and 2005),encoding coiled-coil nucleotide binding leucine-rich repeat (CC-NB-LRR)proteins orthologous to the barley Mla gene cluster, that providesresistance to Blumeria graminis f. sp. hordei (powdery mildew pathogen)(Wei et al., 1999 and 2002). However, it was not known which if any ofthese proteins was the product of the Sr50 gene itself. The recentlycloned Sr33 gene from Triticum tauschii (Periyannan et al., 2013) is anortholog of Mla, and like Sr50 provides resistance to worldwide Pgtisolates so again it has not been possible to distinguish these twospecificities.

The Mla locus of barley is one of the most diverse classes of resistancegenes in cereals, encoding more than 30 different alleles with differentresistance specificities to barley powdery mildew (Seeholzer et al.,2010). This locus is also a potential source of useful diseaseresistance in other cereals, including wheat. For instance, the wheatTmMla1 gene present in the diploid A-genome wheat species Triticummonococcum is an ortholog of Mla and confers race-specific resistance towheat powdery mildew (Jordan et al., 2011). The recently identified Sr33gene, transferred to wheat from the diploid D-genome species Ae.tauschii, is the first known member of the Mla family to provideresistance to Pgt (Periyannan et al., 2013).

There is an urgent need for the identification of genes which confer atleast some level of resistance to plants, especially wheat, againstPuccinia graminis, such as the Ug99 group of races of Puccinia graminisf. sp. tritici.

SUMMARY OF THE INVENTION

The present inventors have identified polypeptides which confer at leastsome level of resistance to plants, especially rye and wheat, againstPuccinia graminis, such as the Ug99 group of races of Puccinia graminisf. sp. tritici.

Thus, in a first aspect the present invention provides a transgenicplant which has integrated into its genome an exogenous polynucleotideencoding a polypeptide which confers resistance to Puccinia graminis,wherein the polynucleotide is operably linked to a promoter capable ofdirecting expression of the polynucleotide in a cell of the plant.

In an embodiment, the Puccinia graminis is Puccinia graminis f. sp.tritici. In a further embodiment, the Puccinia graminis f. sp. triticiis a race of the Ug99 group.

In another embodiment, the transgenic plant has enhanced resistance toPuccinia graminis when compared to an isogenic plant lacking theexogenous polynucleotide.

In an embodiment, the polypeptide is an Sr50 polypeptide.

In a further embodiment,

i) the polypeptide comprises amino acids having a sequence as providedin SEQ ID NO:1, a biologically active fragment thereof, or an amino acidsequence which is at least 82% identical to SEQ ID NO:1, and/or

ii) the polynucleotide comprises nucleotides having a sequence asprovided in SEQ ID NO:10, a sequence which is at least 82% identical toSEQ ID NO:10, or a sequence which hybridizes to SEQ ID NO:10.

In an embodiment, the polypeptide comprises one or more, preferably all,of a coiled coil (CC) domain, an nucleotide binding (NB) domain and aleucine rich repeat (LRR) domain.

In a further embodiment, the polypeptide comprises one or more,preferably all, of a p-loop motif, and a kinase3a motif in the NBdomain.

In an embodiment, the p-loop motif comprises the sequence GxxGxGK(T/S)T(SEQ ID NO:4), more preferably the sequence GFGGLGKTT (SEQ ID NO:5).

In an embodiment, the kinase 3a motif comprises the sequence GxxxxxTxR(SEQ ID NO:6), more preferably the sequence GSRLITTTR (SEQ ID NO:7).

In a further embodiment, the LRR domain comprises about 5 to about 20,or about 10 to about 20, imperfect repeats of the sequence xxLxLxxxx(SEQ ID NO:8).

In an embodiment, the polypeptide confers greater resistance to Pucciniagraminis f. sp. tritici race TTKSK than Sr33 (with a sequence of aminoacids as provided in SEQ ID NO:13). In another embodiment, Sr33 (with asequence of amino acids as provided in SEQ ID NO:13) confers greaterresistance to Puccinia graminis f. sp. tritici race QFCSC than apolypeptide of the invention. In an embodiment, as detailed in Example2, the greater resistance is determined when the polypeptide of theinvention is in T. aestivum line Gabo 1DL.1RS-DR.A1 and when Sr33 is inT. aestivum line Westonia/CS1D5405.

Preferably, the plant is a cereal plant. Examples of transgenic cerealplants of the invention include, but are not limited to wheat, barley,maize, rice, oats, sorghum and triticale. In a particularly preferredembodiment, the plant is wheat.

In a further embodiment, the plant comprises one or more furtherexogenous polynucleotides encoding another plant pathogen resistancepolypeptide. Examples of such other plant pathogen resistancepolypeptides include, but are not limited to, Lr34, Lr1, Lr3, Lr2a,Lr3ka, Lr11, Lr13, Lr16, Lr17, Lr18, Lr21, LrB, Sr35 and Sr33. In anembodiment, the plant at least further comprises an exogenouspolynucleotide encoding plant pathogen resistance polypeptide Sr33. Forexample, the at least further comprises an exogenous polynucleotideencoding a polypeptide comprising amino acids having a sequence asprovided in SEQ ID NO:13 or SEQ ID NO:14, or an amino acid sequencewhich is at least 87% identical, at least 90% identical, or at least 95%identical, to one or both of SEQ ID NO:13 and SEQ ID NO:14 which confersresistance to Puccinia graminis.

Preferably, the plant is homozygous for the exogenous polynucleotide.

In an embodiment, the plant is growing in a field.

In a further aspect, the present invention provides a transgenic plantwhich has integrated into its genome an exogenous polynucleotideencoding a polypeptide which comprises amino acids having a sequence asprovided in SEQ ID NO:1, or an amino acid sequence which is at least 82%identical to SEQ ID NO:1, wherein the polynucleotide is operably linkedto a promoter capable of directing expression of the polynucleotide in acell of the plant.

Also provided is a population of at least 100 transgenic plants of theinvention growing in a field.

In a further aspect, the present invention provides a process foridentifying a polynucleotide encoding a polypeptide which confersresistance to Puccinia graminis comprising:

i) obtaining a polynucleotide operably linked to a promoter, thepolynucleotide encoding a polypeptide comprising amino acids having asequence as provided in SEQ ID NO:1, a biologically active fragmentthereof, or an amino acid sequence which is at least 82% identical toSEQ ID NO:1,

ii) introducing the polynucleotide into a plant,

iii) determining whether the level of resistance to Puccinia graminis ismodified relative to an isogenic plant lacking the polynucleotide, and

iv) optionally, selecting a polynucleotide which when expressed confersresistance to Puccinia graminis.

In an embodiment the process has one or more of the following,

a) the polynucleotide comprises nucleotides having a sequence asprovided in SEQ ID NO:10, a sequence which is at least 82% identical toSEQ ID NO:10, or a sequence which hybridizes to SEQ ID NO:10,

b) the plant is a cereal plant such as a wheat plant,

c) the polypeptide is a plant polypeptide or mutant thereof, and

d) step ii) further comprises stably integrating the polynucleotideoperably linked to a promoter into the genome of the plant.

Also provided is a substantially purified and/or recombinant Pucciniagraminis plant resistance polypeptide.

In an embodiment, the polypeptide is an Sr50 polypeptide.

In another embodiment, the polypeptide comprises amino acids having asequence as provided in SEQ ID NO:1, a biologically active fragmentthereof, or an amino acid sequence which is at least 82% identical, atleast 90% identical, or at least 95% identical, to SEQ ID NO:1.

In a further aspect, the present invention provides a substantiallypurified and/or recombinant polypeptide comprising amino acids having asequence as provided in SEQ ID NO:1, or an amino acid sequence which isat least 82% identical, at least 90% identical, or at least 95%identical, to SEQ ID NO:1.

In an embodiment, a polypeptide of the invention is a fusion proteinfurther comprising at least one other polypeptide sequence. The at leastone other polypeptide may be, for example, a polypeptide that enhancesthe stability of a polypeptide of the present invention, or apolypeptide that assists in the purification or detection of the fusionprotein.

In yet a further aspect, the present invention provides an isolatedand/or exogenous polynucleotide comprising nucleotides having a sequenceas provided in SEQ ID NO:10, a sequence which is at least 82% identicalto SEQ ID NO:10, a sequence encoding a polypeptide of the invention, ora sequence which hybridizes to SEQ ID NO:10.

In another aspect, the present invention provides a chimeric vectorcomprising the polynucleotide of the invention.

Preferably, the polynucleotide is operably linked to a promoter.

In a further aspect, the present invention provides a recombinant cellcomprising an exogenous polynucleotide of the invention and/or a vectorof the invention.

The cell can be any cell type such as, but not limited to, a plant cell,a bacterial cell, an animal cell or a yeast cell.

Preferably, the cell is a plant cell. More preferably, the plant cell isa cereal plant cell. Even more preferably, the cereal plant cell is awheat cell.

In a further aspect, the present invention provides a method ofproducing the polypeptide of the invention, the method comprisingexpressing in a cell or cell free expression system the polynucleotideof the invention.

Preferably, the method further comprises isolating the polypeptide.

In yet another aspect, the present invention provides a transgenicnon-human organism comprising an exogenous polynucleotide of theinvention, a vector of the invention and/or a recombinant cell of theinvention.

Preferably, the transgenic non-human organism is a plant. Preferably,the plant is a cereal plant. More preferably, the cereal plant is awheat plant.

In another aspect, the present invention provides a method of producingthe cell of the invention, the method comprising the step of introducingthe polynucleotide of the invention, or a vector of the invention, intoa cell.

Preferably, the cell is a plant cell.

In a further aspect, the present invention provides a method ofproducing a transgenic plant of the invention, the method comprising thesteps of

i) introducing a polynucleotide of the invention and/or a vector of theinvention into a cell of a plant,

ii) regenerating a transgenic plant from the cell, and

iii) optionally harvesting seed from the plant, and/or

iv) optionally producing one or more progeny plants from the transgenicplant, thereby producing the transgenic plant.

In a further aspect, the present invention provides a method ofproducing a plant which has integrated into its genome a polynucleotideencoding a polypeptide which confers resistance to Puccinia graminis,the method comprising the steps of

i) crossing two parental plants, wherein at least one plant comprises apolynucleotide encoding a polypeptide which confers resistance toPuccinia graminis,

ii) screening one or more progeny plants from the cross for the presenceor absence of the polynucleotide, and

iii) selecting a progeny plant which comprise the polynucleotide,thereby producing the plant.

In an embodiment, at least one of the parental plants is a transgenicplant of the invention, and the selected progeny plant comprises anexogenous polynucleotide encoding a polypeptide which confers resistanceto Puccinia graminis.

In a further embodiment, at least one of the parental plants is atetraploid or hexaploid wheat plant.

In yet another embodiment, step ii) comprises analysing a samplecomprising DNA from the plant for the polynucleotide.

In another embodiment, step iii) comprises

i) selecting progeny plants which are homozygous for the polynucleotide,and/or

ii) analysing the plant or one or more progeny plants thereof forresistance to Puccinia graminis.

In an embodiment, the method further comprises

iv) backcrossing the progeny of the cross of step i) with plants of thesame genotype as a first parent plant which lacked a polynucleotideencoding a polypeptide which confers resistance to Puccinia graminis fora sufficient number of times to produce a plant with a majority of thegenotype of the first parent but comprising the polynucleotide, and

iv) selecting a progeny plant which has resistance to Puccinia graminis.

In yet another aspect, a method of the invention further comprises thestep of analysing the plant for at least one other genetic marker.

Also provide is a plant produced using a method of the invention.

In another aspect, the present invention provides for the use of thepolynucleotide of the invention, or a vector of the invention, toproduce a recombinant cell and/or a transgenic plant.

In an embodiment, the transgenic plant has enhanced resistance toPuccinia graminis when compared to an isogenic plant lacking theexogenous polynucleotide and/or vector.

In a further aspect, the present invention provides a method foridentifying a plant comprising a polynucleotide encoding a polypeptidewhich confers resistance to Puccinia graminis, the method comprising thesteps of

i) obtaining a nucleic acid sample from a plant, and

ii) screening the sample for the presence or absence of thepolynucleotide, wherein presence of the polynucleotide indicates thatthe plant is resistant to Puccinia graminis.

In an embodiment, the polynucleotide encodes a polypeptide of theinvention.

In a further embodiment, the method identifies a transgenic plant of theinvention.

In another embodiment, the method further comprises producing a plantfrom a seed before step i).

Also provided is a plant part of the plant of the invention.

In an embodiment, the plant part is a seed that comprises an exogenouspolynucleotide which encodes a polypeptide which confers resistance toPuccinia graminis.

In a further aspect, the present invention provides a method ofproducing a plant part, the method comprising,

a) growing a plant of the invention, and

b) harvesting the plant part.

In another aspect, the present invention provides a method of producingflour, wholemeal, starch or other product obtained from seed, the methodcomprising;

a) obtaining seed of the invention, and

b) extracting the flour, wholemeal, starch or other product.

In a further aspect, the present invention provides a product producedfrom a plant of the invention and/or a plant part of the invention.

In an embodiment, the part is a seed.

In an embodiment, the product is a food product or beverage product.Examples include, but are not limited to;

i) the food product being selected from the group consisting of: flour,starch, leavened or unleavened breads, pasta, noodles, animal fodder,breakfast cereals, snack foods, cakes, malt, beer, pastries and foodscontaining flour-based sauces, or

ii) the beverage product being beer or malt.

In an alternative embodiment, the product is a non-food product.Examples include, but are not limited to, films, coatings, adhesives,building materials and packaging materials.

In a further aspect, the present invention provides a method ofpreparing a food product of the invention, the method comprising mixingseed, or flour, wholemeal or starch from the seed, with another foodingredient.

In another aspect, the present invention provides a method of preparingmalt, comprising the step of germinating seed of the invention.

Also provided is the use of a plant of the invention, or part thereof,as animal feed, or to produce feed for animal consumption or food forhuman consumption.

In a further aspect, the present invention provides a compositioncomprising one or more of a polypeptide of the invention, apolynucleotide of the invention, a vector of the invention, or arecombinant cell of the invention, and one or more acceptable carriers.

In another aspect, the present invention provides a method ofidentifying a compound that binds to a polypeptide comprising aminoacids having a sequence as provided in SEQ ID NO:1, a biologicallyactive fragment thereof, or an amino acid sequence which is at least 82%identical to SEQ ID NO:1, the method comprising:

i) contacting the polypeptide with a candidate compound, and

ii) determining whether the compound binds the polypeptide.

Any embodiment herein shall be taken to apply mutatis mutandis to anyother embodiment unless specifically stated otherwise.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended for the purpose ofexemplification only. Functionally-equivalent products, compositions andmethods are clearly within the scope of the invention, as describedherein.

Throughout this specification, unless specifically stated otherwise orthe context requires otherwise, reference to a single step, compositionof matter, group of steps or group of compositions of matter shall betaken to encompass one and a plurality (i.e. one or more) of thosesteps, compositions of matter, groups of steps or group of compositionsof matter.

The invention is hereinafter described by way of the followingnon-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1 . Isolation of phage lambda DNA clones from the Sr50 locus. Theautoradiograph shows hybridisation of P32-labelled probe B76 to DraIdigested lambda clones 4, 5, 8, 11, 12, and 18 and genomic DNA of wheatplants Gabo 1BL.1RS. The gel-purified region used to make the lambdagenomic DNA library is indicated on the Gabo1BL.1RS lane in a dotted boxwith the fragments missing in deletion mutants indicated by arrows.

FIG. 2 . Schematic representation of the BAC contig at the Sr50 locus.The map of five overlapping BAC clones is shown, spanning the deletion(dotted line) in mutant M2. BAC end sequences within the deletion werenot present in M2, whereas BAC end sequences outside of the deletionwere present in M2. The relative positions of ScRGA1-A to G gene familymembers and of a predicted chymotrypsin inhibitor gene (ScCI2) are shownwithin the five BAC clones. ScRGA1-A was shown to be the Sr50 gene asdescribed herein.

FIG. 3 . Structure of the Sr50 gene. The Figure shows a schematicrepresentation of the structure of the Sr50 (ScRGA1-A) gene includingthe 5′ and 3′ UTRs, the sizes of introns and exons (in basepairs; bp)and position of mutations in mutants M7 and M13. The relative positionsof the CC, NB and LRR domains in the Sr50 polypeptide and the positionof the 5pF3 and 5pR2 primers for amplification of a region including thetranslational start codon are also shown.

FIG. 4 . Phylogenetic relationship of Sr50 and related Mla familyCC-NB-LRR proteins. A neighbor-joining tree was obtained from thepredicted amino acid sequences of ScRGA1 genes from S. cereale, knownfunctional MLA members of barley (HvMLA), TmMLA from T. monococcum,Sr33, Sr35 and leaf rust resistance polypeptides Lr1, Lr10 and Lr21.

KEY TO THE SEQUENCE LISTING

SEQ ID NO:1—Amino acid sequence of stem rust resistance polypeptide(Sr50).SEQ ID NO:2—Amino acid sequence of Sr50 variant from Svalofs Otello andFrontier.SEQ ID NO:3—Amino acid sequence of Sr50 variant from Dwarf Petkus.SEQ ID NO:4—Consenus p-loop motif.SEQ ID NO:5—P-loop motif of polypeptide provided as SEQ ID NO:1.SEQ ID NO:6—Consenus kinase 3a motif.SEQ ID NO:7—Kinase 3a motif of polypeptide provided as SEQ ID NO:1.SEQ ID NO:8—Consensus repeat of the LRR domain.SEQ ID NO:9—Nucleotide sequence of cDNA encoding stem rust resistancepolypeptide (Sr50).SEQ ID NO:10—Nucleotide sequence of open reading frame encoding stemrust resistance polypeptide (Sr50).SEQ ID NO:11—Nucleotide sequence of pVecNeoSr50 expression construct.SEQ ID NO:12—Nucleotide sequence of pVecBarSr50 expression construct.SEQ ID NO:13—Amino acid sequence of stem rust resistance polypeptide(from haplotype I) (Sr33).SEQ ID NO:14—Amino acid sequence of allelic variant of the stem rustresistance polypeptide provided as SEQ ID NO:13 (from haplotype II)(Sr33).SEQ ID NO:15—Functional nuclear export signal NES from HIV Rev.

SEQ ID NO:16—Non-functional nes.

SEQ ID NOs 17 to 59—Oligonucleotide primers.

DETAILED DESCRIPTION OF THE INVENTION General Techniques and Definitions

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (e.g., in cell culture,molecular genetics, plant molecular biology, protein chemistry, andbiochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, andimmunological techniques utilized in the present invention are standardprocedures, well known to those skilled in the art. Such techniques aredescribed and explained throughout the literature in sources such as, J.Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons(1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbour Laboratory Press (1989), T. A. Brown (editor), EssentialMolecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press(1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A PracticalApproach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel etal. (editors), Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience (1988, including all updates untilpresent), Ed Harlow and David Lane (editors) Antibodies: A LaboratoryManual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al.(editors) Current Protocols in Immunology, John Wiley & Sons (includingall updates until present).

The term “and/or”, e.g., “X and/or Y” shall be understood to mean either“X and Y” or “X or Y” and shall be taken to provide explicit support forboth meanings or for either meaning.

As used herein, the term about, unless stated to the contrary, refers to+/−10%, more preferably +/−5%, more preferably +/−1%, more preferably+/−0.5%, of the designated value.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

Stem Rust

As used herein, “stem rust” refers to the disease of plants caused byPuccinia graminis or to the causative fungal pathogen, Pucciniagraminis, as the context determines. As used herein, “wheat stem rust”refers to the disease of plants caused by Puccinia graminis f. sp.tritici or to the causative fungal pathogen, Puccinia graminis f. sp.tritici, as the context determines.

The Ug99 group of races of wheat stem rust (Puccinia graminis f. sp.tritici) (also known as ‘TTKSK’ under the North American nomenclaturesystem) is a well known fungal pathogen of wheat and is commonly presentin wheat fields in countries such as in Africa and the Middle East(Singh et al., 2011; Hodson et al., 2012). Ug99 can cause major croplosses and is virulent against resistance genes that have previouslyprotected wheat against stem rust. There are currently eight knownvariants of group Ug99 which are closely related based on DNA markeranalysis. Each variant of the pathogen which differs in itsvirulence/avirulence profile on a panel of wheat plants each comprisinga different resistance R gene is known as a “race” of the pathogen. TheUg99 group of isolates are all closely related and are believed to haveevolved from a common ancestor, but may differ in theirvirulence/avirulence profiles in which case they are considereddifferent races. Seven of these eight variants are summarized in Table 2of Singh et al. (2011). In an embodiment, the Ug99 group of stem rustraces exhibit virulence on wheat plants comprising one or more of theresistance genes Sr31, Sr21, Sr24 and Sr36 (Singh et al., 2011). In oneembodiment, the Ug99 group of stem rust races of Puccinia graminis f.sp. tritici has virulence at least to wheat plants comprising theresistance gene Sr31 (Pretorius et al., 2000).

Polypeptides/Peptides

The present invention relates to polypeptides which confer resistance toa plant, for example a wheat plant, to stem rust, preferably to wheatstem rust such as the Ug99 group of races. In a preferred embodiment,the polypeptide is encoded by an allele or variant of an Sr50 gene whichconfers resistance to wheat stem rust. Examples of such polypeptidesinclude, but are not limited to, those comprising an amino acid sequenceas provided in SEQ ID NO:1. The polypeptide of the invention confersenhanced resistance to stem rust, preferably wheat stem rust such as theUg99 group of races of Puccinia graminis f. sp. tritici when compared toan isogenic plant lacking a gene encoding the polypeptide. This termalso refers to the naturally produced protein (or wild-type protein fromwhich a mutant protein is derived) encoded by a gene conferring upon aplant (for example, wheat), when grown in normal field conditions,enhanced resistance to stem rust such as the Ug99 group of races ofPuccinia graminis f. sp. tritici. In a preferred embodiment, thepolypeptide of the invention confers resistance specifically to stemrust, preferably specifically to wheat stem rust, more preferably itdoes not confer resistance to wheat leaf rust caused by the fungalpathogen Puccinia triticina and/or to powdery mildew. In this context,“specifically to stem rust” and “specifically to wheat stem rust” meansthat the conferred resistance is preferentially to stem rust or wheatstem rust in comparison to another fungal pathogen of the same plantspecies, preferably to many or most other fungal pathogens of the samespecies. In a more preferred embodiment, the polypeptide of theinvention confers resistance to stem rust and at least two, or allthree, of leaf rust, stripe rust and powdery mildew, preferably inwheat. In an embodiment, polypeptides of the invention are not encodedby the Sr35 gene of a wheat plant. In an embodiment, polypeptides of theinvention are not encoded by the Sr35 gene of a wheat plant or itshomologs, such as those that are at least 50% identical in amino acidsequence to the Sr35 polypeptide. In another embodiment, polypeptides ofthe invention are not encoded by the Sr33 gene of a wheat plant. In anembodiment, polypeptides of the invention are not encoded by the Sr33gene of a wheat plant or its homologs, such as those that are at least87% identical in amino acid sequence to the Sr33 polypeptide asdescribed in WO 2014/000594 (SEQ ID NO's 13 and 14 herein). Thus, in anembodiment, a polypeptide of the invention does not comprise amino acidshaving a sequence at least 87% identical in amino acid sequence to SEQID NO:13 and/or SEQ ID NO:14. In a further embodiment, a polypeptide ofthe invention has an amino acid sequence which is more closely related(has a higher % identity level) to SEQ ID NO:1 than SEQ ID NO:13 and/or14.

In a further embodiment, when expressed in a transgenic plant infectedwith stem rust, such as with a Ug99 race of Puccinia graminis f. sp.tritici, the cells of the plant display little, if any, signs of celldeath (autofluorescence), for instance when compared to an isogenicplant expressing Sr45.

Polypeptides of the invention typically comprise a coiled coil (CC)domain towards the N-terminus, followed by an nucleotide binding (NB)domain and a leucine rich repeat (LRR) domain towards the C-terminus(see FIG. 3 ). Each of these three types of domains are common inpolypeptides that confer resistance to plant pathogens. In addition,CC-NB-LRR containing polypeptides are a known large class ofpolypeptides which, as a class, confer resistance across a wide varietyof different plant pathogens (see, for example, Bulgarelli et al., 2010;McHale et al., 2006; Takken et al., 2006; Wang et al., 2011; Gennaro etal., 2009; and Dilbirligi et al., 2003), although each CC-NB-LRRpolypeptides is specific to a particular species or sub-species ofpathogen. Accordingly, by aligning the polypeptides of the inventionwith other CC-NB-LRR polypeptides, combined with the large number ofstudies on these types of proteins as well as CC domains, NB domains andLRR domains, the skilled person has a considerable amount of guidancefor designing functional variants of the specific polypeptides providedherein.

A coiled-coil domain or motif is a structural motif which is one of themost common tertiary structures of proteins where a-helices are coiledtogether like the strands of a rope. Computer programs have been devisedto detect heptads and resulting in coiled-coil structures (see, forexample Delorenzi and Speed, 2002). Coiled coils typically comprise arepeated pattern, hxxhcxc, of hydrophobic (h) and charged (c) amino-acidresidues, referred to as a heptad repeats. The positions in the heptadrepeat are usually labeled abcdefg, where a and d are the hydrophobicpositions, often being occupied by isoleucine, alanine, leucine orvaline. Folding a protein with these hepatds into an a-helical secondarystructure causes the hydrophobic residues to be presented as a ‘stripe’that coils gently around the helix in left-handed fashion, forming anamphipathic structure.

The NB domain is present in resistance genes as well as several kinasessuch as ATP/GTP-binding proteins. This domain typically contains threemotifs: kinase-la (p-loop), a kinase-2, and a putative kinase-3a (Traut1994; Tameling et al., 2002). The consensus sequence of GxxGxGK(T/S)T(SEQ ID NO:4) (GFGGLGKTT (SEQ ID NO:5) in the polypeptide which confersresistance to Puccinia graminis provided as SEQ ID NO:1), and GxxxxxTxR(SEQ ID NO:6) (GSRLITTTR (SEQ ID NO:7) in the polypeptide which confersresistance to Puccinia graminis provided as SEQ ID NO:1) for theresistance gene motifs p-loop, kinase-2, and the putative kinase-3a,respectively, are different from those present in other NB-encodingproteins. Other motifs present in the NB domain of NB/LRR-typeresistance genes are GLPL, RNBS-D and MHD (Meyers et al., 1999). Thesequences interspersing these motifs and domains can be very differenteven among homologues of a resistance gene (Michelmore and Meyers, 1998;Pan et al., 2000).

A leucine-rich domain is a protein structural motif that forms an α/βhorseshoe fold (Enkhbayar et al., 2004). The LRR domain contains 9-41imperfect repeats, each about 25 amino acids long with a consensus aminoacid sequence of xxLxLxxxx (SEQ ID NO:8) (Cooley et al., 2000). In anembodiment, a polypeptide of the invention comprises about 10 to about20, more preferably about 12 to about 18, more preferably about 15leucine rich repeats. These repeats commonly fold together to form asolenoid protein domain Typically, each repeat unit has betastrand-turn-alpha helix structure, and the assembled domain, composed ofmany such repeats, has a horseshoe shape with an interior parallel betasheet and an exterior array of helices.

In an embodiment, the polypeptide comprises one, two, three, four ormore amino acids which are present in the amino acid sequence providedas SEQ ID NO:1 but which are not found in the corresponding amino acidposition of a polypeptide comprising amino acids having a sequence asprovided in SEQ ID NO:13 and SEQ ID NO:14.

In an embodiment, the polypeptide does not comprise amino acids having asequence as provided in SEQ ID NO:1 or SEQ ID NO:2, a biologicallyactive fragment thereof, or an amino acid sequence which is at least 87%identical to one or both of SEQ ID NO:13 and SEQ ID NO:14. In anembodiment, polypeptide does not comprise amino acids having a sequenceas provided in SEQ ID NO:13 or SEQ ID NO:14.

In an embodiment, the polypeptide is from S. cereale.

As used herein, “resistance” is a relative term in that the presence ofa polypeptide of the invention (i) reduces the disease symptoms of aplant comprising the gene (R gene) that confers resistance, relative toa plant lacking the R gene, and/or (ii) reduces pathogen reproduction orspread on a plant comprising the R gene. Resistance as used herein isrelative to the “susceptible” response of a plant to the same pathogen.Typically, the presence of the R gene improves at least one productiontrait of a plant comprising the R gene when infected with the pathogen,such as grain yield, when compared to an isogenic plant infected withthe pathogen but lacking the R gene. The isogenic plant may have somelevel of resistance to the pathogen, or may be classified assusceptible. Thus, the terms “resistance” and “enhanced resistance” aregenerally used herein interchangeably. Furthermore, a polypeptide of theinvention does not necessarily confer complete pathogen resistance, forexample when some symptoms still occur or there is some pathogenreproduction on infection but at a reduced amount Enhanced resistancecan be determined by a number of methods known in the art such asanalysing the plants for the amount of pathogen and/or analysing plantgrowth or the amount of damage or disease symptoms to a plant in thepresence of the pathogen, and comparing one or more of these parametersto an isogenic plant lacking an exogenous gene encoding a polypeptide ofthe invention.

By “substantially purified polypeptide” or “purified polypeptide” wemean a polypeptide that has generally been separated from the lipids,nucleic acids, other peptides, and other contaminating molecules withwhich it is associated in its native state. Preferably, thesubstantially purified polypeptide is at least 90% free from othercomponents with which it is naturally associated.

Transgenic plants and host cells of the invention may comprise anexogenous polynucleotide encoding a polypeptide of the invention. Inthese instances, the plants and cells produce a recombinant polypeptide.The term “recombinant” in the context of a polypeptide refers to thepolypeptide encoded by an exogenous polynucleotide when produced by acell, which polynucleotide has been introduced into the cell or aprogenitor cell by recombinant DNA or RNA techniques such as, forexample, transformation. Typically, the cell comprises a non-endogenousgene that causes an altered amount of the polypeptide to be produced. Inan embodiment, a “recombinant polypeptide” is a polypeptide made by theexpression of an exogenous (recombinant) polynucleotide in a plant cell.

The terms “polypeptide” and “protein” are generally usedinterchangeably.

The % identity of a polypeptide is determined by GAP (Needleman andWunsch, 1970) analysis (GCG program) with a gap creation penalty=5, anda gap extension penalty=0.3. The query sequence is at least 150 aminoacids in length, and the GAP analysis aligns the two sequences over aregion of at least 150 amino acids. More preferably, the query sequenceis at least 500 amino acids in length, and the GAP analysis aligns thetwo sequences over a region of at least 500 amino acids. Morepreferably, the query sequence is at least 750 amino acids in length andthe GAP analysis aligns the two sequences over a region of at least 750amino acids. Even more preferably, the query sequence is at least 900amino acids in length and the GAP analysis aligns the two sequences overa region of at least 900 amino acids. Even more preferably, the GAPanalysis aligns two sequences over their entire length.

As used herein a “biologically active” fragment is a portion of apolypeptide of the invention which maintains a defined activity of thefull-length polypeptide such as when expressed in a plant, such aswheat, confers (enhanced) resistance to stem rust, preferably wheat stemrust such as the Ug99 group of races of Puccinia graminis f. sp. triticiwhen compared to an isogenic plant not expressing the polypeptide.Biologically active fragments can be any size as long as they maintainthe defined activity but are preferably at least 750 or at least 900amino acid residues long. Preferably, the biologically active fragmentmaintains at least 10%, at least 50%, at least 75% or at least 90%, ofthe activity of the full length protein.

With regard to a defined polypeptide, it will be appreciated that %identity figures higher than those provided above will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polypeptide comprises anamino acid sequence which is at least 60%, more preferably at least 65%,more preferably at least 70%, more preferably at least 75%, morepreferably at least 76%, more preferably at least 80%, more preferablyat least 85%, more preferably at least 90%, more preferably at least91%, more preferably at least 92%, more preferably at least 93%, morepreferably at least 94%, more preferably at least 95%, more preferablyat least 96%, more preferably at least 97%, more preferably at least98%, more preferably at least 99%, more preferably at least 99.1%, morepreferably at least 99.2%, more preferably at least 99.3%, morepreferably at least 99.4%, more preferably at least 99.5%, morepreferably at least 99.6%, more preferably at least 99.7%, morepreferably at least 99.8%, and even more preferably at least 99.9%identical to the relevant nominated SEQ ID NO.

Amino acid sequence mutants of the polypeptides of the present inventioncan be prepared by introducing appropriate nucleotide changes into anucleic acid of the present invention, or by in vitro synthesis of thedesired polypeptide. Such mutants include, for example, deletions,insertions or substitutions of residues within the amino acid sequence.A combination of deletion, insertion and substitution can be made toarrive at the final construct, provided that the final peptide productpossesses the desired characteristics. Preferred amino acid sequencemutants have only one, two, three, four or less than 10 amino acidchanges relative to the reference wildtype polypeptide.

Mutant (altered) polypeptides can be prepared using any technique knownin the art, for example, using directed evolution or rational designstrategies (see below). Products derived from mutated/altered DNA canreadily be screened using techniques described herein to determine ifthey confer resistance to Puccinia graminis (for example, a race of theUg99 group of Puccinia graminis f sp. tritici) such as by producing atransgenic plant expressing the mutated/altered DNA and determining theability of the plant to produce grain in the presence of the pathogen.

In designing amino acid sequence mutants, the location of the mutationsite and the nature of the mutation will depend on characteristic(s) tobe modified. The sites for mutation can be modified individually or inseries, e.g., by (1) substituting first with conservative amino acidchoices and then with more radical selections depending upon the resultsachieved, (2) deleting the target residue, or (3) inserting otherresidues adjacent to the located site.

Amino acid sequence deletions generally range from about 1 to 15residues, more preferably about 1 to 10 residues and typically about 1to 5 contiguous residues.

Substitution mutants have at least one amino acid residue in thepolypeptide molecule removed and a different residue inserted in itsplace. In order to maintain activity, sites of interest include thosenot in an active site, such as a CC, BD or LRR domain, and those whichare not highly conserved between different species. These sites,especially those falling within a sequence of at least three othernon-conserved sites can generally be substituted in a relativelyconservative or non-conservative manner Examples of conservativesubstitutions are shown in Table 1 under the heading of “exemplarysubstitutions”.

TABLE 1 Exemplary substitutions. Original Residue ExemplarySubstitutions Ala (A) val; leu; ile; gly Arg (R) lys Asn (N) gln; hisAsp (D) glu Cys (C) ser Gln (Q) asn; his Glu (E) asp Gly (G) pro, alaHis (H) asn; gln Ile (I) leu; val; ala Leu (L) ile; val; met; ala; pheLys (K) arg Met (M) leu; phe Phe (F) leu; val; ala Pro (P) gly Ser (S)thr Thr (T) ser Trp (W) tyr Tyr (Y) trp; phe Val (V) ile; leu; met; phe,ala

In a preferred embodiment a mutant/variant polypeptide has one or two orthree or four conservative amino acid changes when compared to anaturally occurring polypeptide. Details of conservative amino acidchanges are provided in Table 1. In a preferred embodiment, the changesare not in one or more of the motifs which are highly conserved betweenthe different polypeptides provided herewith. As the skilled personwould be aware, such minor changes can reasonably be predicted not toalter the activity of the polypeptide when expressed in a recombinantcell.

In an embodiment, the protein of the invention is a CC-NB-LRR plantpathogen resistance gene which comprises domains configured as shown inFIG. 3 .

The primary amino acid sequence of a polypeptide of the invention can beused to design variants/mutants thereof based on comparisons withclosely related resistance polypeptides comprising NB and LRR domains,more preferably CC, NB and LRR domains. As the skilled addressee willappreciate, residues highly conserved amongst closely related CC-NB-LRRproteins are less likely to be able to be altered, especially withnon-conservative substitutions, and activity maintained than lessconserved residues (see above).

Also included within the scope of the invention are polypeptides of thepresent invention which are differentially modified during or aftersynthesis, e.g., by biotinylation, benzylation, glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to an antibodymolecule or other cellular ligand, etc. The polypeptides may bepost-translationally modified in a cell, for example by phosphorylation,which may modulate its activity. These modifications may serve toincrease the stability and/or bioactivity of the polypeptide of theinvention.

Directed Evolution

In directed evolution, random mutagenesis is applied to a protein, and aselection regime is used to pick out variants that have the desiredqualities, for example, increased activity. Further rounds of mutationand selection are then applied. A typical directed evolution strategyinvolves three steps:

1) Diversification: The gene encoding the protein of interest is mutatedand/or recombined at random to create a large library of gene variants.Variant gene libraries can be constructed through error prone PCR (see,for example, Leung, 1989, Cadwell and Joyce, 1992), from pools of DNaseldigested fragments prepared from parental templates (Stemmer, 1994a;Stemmer, 1994b: Crameri et al., 1998; Coco et al., 2001) from degenerateoligonucleotides (Ness et al., 2002, Coco, 2002) or from mixtures ofboth, or even from undigested parental templates (Zhao et al., 1.998;Eggert et al., 2005; Jézéquek et al, 2008) and are usually assembledthrough PCR. Libraries can also be made from parental sequencesrecombined in vivo or in vitro by either homologous or non-homologousrecombination (Ostermeier et al., 1999; Volkov et al., 1999; Sieber etal., 2001). Variant gene libraries can also be constructed bysub-cloning a gene of interest into a suitable vector, transforming thevector into a “mutator” strain such as the E. coli XL-1 red (Stratagene)and propagating the transformed bacteria for a suitable number ofgenerations. Variant gene libraries can also be constructed bysubjecting the gene of interest to DNA shuffling (i.e., in vitrohomologous recombination of pools of selected mutant genes by randomfragmentation and reassembly) as broadly described by Harayama (1998).

2) Selection: The library is tested for the presence of mutants(variants) possessing the desired property using a screen or selection.Screens enable the identification and isolation of high-performingmutants by hand, while selections automatically eliminate allnonfunctional mutants. A screen may involve screening for the presenceof known conserved amino acid motifs. Alternatively, or in addition, ascreen may involve expressing the mutated polynucleotide in a hostorganism or part thereof and assaying the level of activity.

3) Amplification: The variants identified in the selection or screen arereplicated many fold, enabling researchers to sequence their DNA inorder to understand what mutations have occurred.

Together, these three steps are termed a “round” of directed evolution.Most experiments will entail more than one round. In these experiments,the “winners” of the previous round are diversified in the next round tocreate a new library. At the end of the experiment, all evolved proteinor polynucleotide mutants are characterized using biochemical methods.

Rational Design

A protein can be designed rationally, on the basis of known informationabout protein structure and folding. This can be accomplished by designfrom scratch (de novo design) or by redesign based on native scaffolds(see, for example, Hellinga, 1997; and Lu and Berry, Protein StructureDesign and Engineering, Handbook of Proteins 2, 1153-1157 (2007)).Protein design typically involves identifying sequences that fold into agiven or target structure and can be accomplished using computer models.Computational protein design algorithms search the sequence-conformationspace for sequences that are low in energy when folded to the targetstructure. Computational protein design algorithms use models of proteinenergetics to evaluate how mutations would affect a protein's structureand function. These energy functions typically include a combination ofmolecular mechanics, statistical (i.e. knowledge-based), and otherempirical terms. Suitable available software includes IPRO (InterativeProtein Redesign and Optimization), EGAD (A Genetic Algorithm forProtein Design), Rosetta Design, Sharpen, and Abalone.

Polynucleotides and Genes

The present invention refers to various polynucleotides. As used herein,a “polynucleotide” or “nucleic acid” or “nucleic acid molecule” means apolymer of nucleotides, which may be DNA or RNA or a combinationthereof, and includes genomic DNA, mRNA, cRNA, and cDNA. Less preferredpolynucleotides include tRNA, siRNA, shRNA and hpRNA. It may be DNA orRNA of cellular, genomic or synthetic origin, for example made on anautomated synthesizer, and may be combined with carbohydrate, lipids,protein or other materials, labelled with fluorescent or other groups,or attached to a solid support to perform a particular activity definedherein, or comprise one or more modified nucleotides not found innature, well known to those skilled in the art. The polymer may besingle-stranded, essentially double-stranded or partly double-stranded.Basepairing as used herein refers to standard basepairing betweennucleotides, including G:U basepairs. “Complementary” means twopolynucleotides are capable of basepairing (hybridizing) along part oftheir lengths, or along the full length of one or both. A “hybridizedpolynucleotide” means the polynucleotide is actually basepaired to itscomplement. The term “polynucleotide” is used interchangeably hereinwith the term “nucleic acid”. Preferred polynucleotides of the inventionencode a polypeptide of the invention.

By “isolated polynucleotide” we mean a polynucleotide which hasgenerally been separated from the polynucleotide sequences with which itis associated or linked in its native state, if the polynucleotide isfound in nature. Preferably, the isolated polynucleotide is at least 90%free from other components with which it is naturally associated, if itis found in nature. Preferably the polynucleotide is not naturallyoccurring, for example by covalently joining two shorter polynucleotidesequences in a manner not found in nature (chimeric polynucleotide).

The present invention involves modification of gene activity and theconstruction and use of chimeric genes. As used herein, the term “gene”includes any deoxyribonucleotide sequence which includes a proteincoding region or which is transcribed in a cell but not translated, aswell as associated non-coding and regulatory regions. Such associatedregions are typically located adjacent to the coding region or thetranscribed region on both the 5′ and 3′ ends for a distance of about 2kb on either side. In this regard, the gene may include control signalssuch as promoters, enhancers, termination and/or polyadenylation signalsthat are naturally associated with a given gene, or heterologous controlsignals in which case the gene is referred to as a “chimeric gene”. Thesequences which are located 5′ of the coding region and which arepresent on the mRNA are referred to as 5′ non-translated sequences. Thesequences which are located 3′ or downstream of the coding region andwhich are present on the mRNA are referred to as 3′ non-translatedsequences. The term “gene” encompasses both cDNA and genomic forms of agene.

A “Sr50 gene” as used herein refers to a nucleotide sequence which ishomologous to the isolated Sr50 gene or Sr50 cDNA (SEQ ID NO:9)described herein. As described herein, some alleles and variants of theSr50 gene family encode a protein that confers resistance to stem rust(for example as caused by the Ug99 group of races of Puccinia graminisf. sp. tritici). Sr50 genes include the naturally occurring alleles orvariants existing in cereals such as wheat. Nucleic acid moleculeshaving the nucleotide sequence shown herein as SEQ ID NO:9 (cDNA) or SEQID NO:10 (open reading frame), encoding a protein with amino acidsequence SEQ ID NO:1, are examples of an Sr50 gene which confersresistance to stem rust.

A genomic form or clone of a gene containing the transcribed region maybe interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences”, which may be eitherhomologous or heterologous with respect to the “exons” of the gene. An“intron” as used herein is a segment of a gene which is transcribed aspart of a primary RNA transcript but is not present in the mature mRNAmolecule. Introns are removed or “spliced out” from the nuclear orprimary transcript; introns therefore are absent in the messenger RNA(mRNA). Introns may contain regulatory elements such as enhancers.“Exons” as used herein refer to the DNA regions corresponding to the RNAsequences which are present in the mature mRNA or the mature RNAmolecule in cases where the RNA molecule is not translated. An mRNAfunctions during translation to specify the sequence or order of aminoacids in a nascent polypeptide. The term “gene” includes a synthetic orfusion molecule encoding all or part of the proteins of the inventiondescribed herein and a complementary nucleotide sequence to any one ofthe above. A gene may be introduced into an appropriate vector forextrachromosomal maintenance in a cell or, preferably, for integrationinto the host genome.

As used herein, a “chimeric gene” refers to any gene that comprisescovalently joined sequences that are not found joined in nature.Typically, a chimeric gene comprises regulatory and transcribed orprotein coding sequences that are not found together in nature.Accordingly, a chimeric gene may comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. The term“endogenous” is used herein to refer to a substance that is normallypresent or produced in an unmodified plant at the same developmentalstage as the plant under investigation. An “endogenous gene” refers to anative gene in its natural location in the genome of an organism. Asused herein, “recombinant nucleic acid molecule”, “recombinantpolynucleotide” or variations thereof refer to a nucleic acid moleculewhich has been constructed or modified by recombinant DNA technology.The terms “foreign polynucleotide” or “exogenous polynucleotide” or“heterologous polynucleotide” and the like refer to any nucleic acidwhich is introduced into the genome of a cell by experimentalmanipulations.

Foreign or exogenous genes may be genes that are inserted into anon-native organism, native genes introduced into a new location withinthe native host, or chimeric genes. A “transgene” is a gene that hasbeen introduced into the genome by a transformation procedure. The term“genetically modified” includes introducing genes into cells bytransformation or transduction, mutating genes in cells and altering ormodulating the regulation of a gene in a cell or organisms to whichthese acts have been done or their progeny.

Furthermore, the term “exogenous” in the context of a polynucleotide(nucleic acid) refers to the polynucleotide when present in a cell thatdoes not naturally comprise the polynucleotide. The cell may be a cellwhich comprises a non-endogenous polynucleotide resulting in an alteredamount of production of the encoded polypeptide, for example anexogenous polynucleotide which increases the expression of an endogenouspolypeptide, or a cell which in its native state does not produce thepolypeptide. Increased production of a polypeptide of the invention isalso referred to herein as “over-expression”. An exogenouspolynucleotide of the invention includes polynucleotides which have notbeen separated from other components of the transgenic (recombinant)cell, or cell-free expression system, in which it is present, andpolynucleotides produced in such cells or cell-free systems which aresubsequently purified away from at least some other components. Theexogenous polynucleotide (nucleic acid) can be a contiguous stretch ofnucleotides existing in nature, or comprise two or more contiguousstretches of nucleotides from different sources (naturally occurringand/or synthetic) joined to form a single polynucleotide. Typically suchchimeric polynucleotides comprise at least an open reading frameencoding a polypeptide of the invention operably linked to a promotersuitable of driving transcription of the open reading frame in a cell ofinterest.

In an embodiment, the polynucleotide is not naturally occurring such ascomprising nucleotides having a sequence as provided in SEQ ID NO:10.For example, in an embodiment the polynucleotide is a codon optimisedpolynucleotide encoding a polypeptide comprising amino acids having asequence as provided in SEQ ID NO:1, a biologically active fragmentthereof, or an amino acid sequence which is at least 82% identical, atleast 90% identical, or at least 95% identical, to SEQ ID NO:1, forexpression in a plant other than rye (such as wheat).

In an embodiment, if present in a rye plant, or part (such a ryegrain)or cell thereof, the polynucleotide is not present on chromosome 1RS.

The % identity of a polynucleotide is determined by GAP (Needleman andWunsch, 1970) analysis (GCG program) with a gap creation penalty=5, anda gap extension penalty=0.3. The query sequence is at least 450nucleotides in length, and the GAP analysis aligns the two sequencesover a region of at least 450 nucleotides. Preferably, the querysequence is at least 1,500 nucleotides in length, and the GAP analysisaligns the two sequences over a region of at least 1,500 nucleotides.Even more preferably, the query sequence is at least 2,700 nucleotidesin length and the GAP analysis aligns the two sequences over a region ofat least 2,700 nucleotides. Even more preferably, the GAP analysisaligns two sequences over their entire length.

With regard to the defined polynucleotides, it will be appreciated that% identity figures higher than those provided above will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polynucleotide comprises apolynucleotide sequence which is at least 60%, more preferably at least65%, more preferably at least 70%, more preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 91%, more preferably at least92%, more preferably at least 93%, more preferably at least 94%, morepreferably at least 95%, more preferably at least 96%, more preferablyat least 97%, more preferably at least 98%, more preferably at least99%, more preferably at least 99.1%, more preferably at least 99.2%,more preferably at least 99.3%, more preferably at least 99.4%, morepreferably at least 99.5%, more preferably at least 99.6%, morepreferably at least 99.7%, more preferably at least 99.8%, and even morepreferably at least 99.9% identical to the relevant nominated SEQ ID NO.

In a further embodiment, the present invention relates topolynucleotides which are substantially identical to those specificallydescribed herein. As used herein, with reference to a polynucleotide theterm “substantially identical” means the substitution of one or a few(for example 2, 3, or 4) nucleotides whilst maintaining at least oneactivity of the native protein encoded by the polynucleotide. Inaddition, this term includes the addition or deletion of nucleotideswhich results in the increase or decrease in size of the encoded nativeprotein by one or a few (for example 2, 3, or 4) amino acids whilstmaintaining at least one activity of the native protein encoded by thepolynucleotide.

The present invention also relates to the use of oligonucleotides, forinstance in methods of screening for a polynucleotide of, or encoding apolypeptide of, the invention. As used herein, “oligonucleotides” arepolynucleotides up to 50 nucleotides in length. The minimum size of sucholigonucleotides is the size required for the formation of a stablehybrid between an oligonucleotide and a complementary sequence on anucleic acid molecule of the present invention. They can be RNA, DNA, orcombinations or derivatives of either. Oligonucleotides are typicallyrelatively short single stranded molecules of 10 to 30 nucleotides,commonly 15-25 nucleotides in length. When used as a probe or as aprimer in an amplification reaction, the minimum size of such anoligonucleotide is the size required for the formation of a stablehybrid between the oligonucleotide and a complementary sequence on atarget nucleic acid molecule. Preferably, the oligonucleotides are atleast 15 nucleotides, more preferably at least 18 nucleotides, morepreferably at least 19 nucleotides, more preferably at least 20nucleotides, even more preferably at least 25 nucleotides in length.Oligonucleotides of the present invention used as a probe are typicallyconjugated with a label such as a radioisotope, an enzyme, biotin, afluorescent molecule or a chemiluminescent molecule.

The present invention includes oligonucleotides that can be used as, forexample, probes to identify nucleic acid molecules, or primers toproduce nucleic acid molecules. Probes and/or primers can be used toclone homologues of the polynucleotides of the invention from otherspecies. Furthermore, hybridization techniques known in the art can alsobe used to screen genomic or cDNA libraries for such homologues.

Polynucleotides and oligonucleotides of the present invention includethose which hybridize under stringent conditions to one or more of thesequences provided as SEQ ID NO's: 9 and/or 10. As used herein,stringent conditions are those that (1) employ low ionic strength andhigh temperature for washing, for example, 0.015 M NaCl/0.0015 M sodiumcitrate/0.1% NaDodSO₄ at 50° C.; (2) employ during hybridisation adenaturing agent such as formamide, for example, 50% (vol/vol) formamidewith 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone,50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodiumcitrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl,0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodiumpyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50g/ml), 0.1% SDS and 10% dextran sulfate at 42° C. in 0.2×SSC and 0.1%SDS.

Polynucleotides of the present invention may possess, when compared tonaturally occurring molecules, one or more mutations which aredeletions, insertions, or substitutions of nucleotide residues. Mutantscan be either naturally occurring (that is to say, isolated from anatural source) or synthetic (for example, by performing site-directedmutagenesis on the nucleic acid). A variant of a polynucleotide or anoligonucleotide of the invention includes molecules of varying sizes of,and/or are capable of hybridising to, the wheat genome close to that ofthe reference polynucleotide or oligonucleotide molecules definedherein. For example, variants may comprise additional nucleotides (suchas 1, 2, 3, 4, or more), or less nucleotides as long as they stillhybridise to the target region. Furthermore, a few nucleotides may besubstituted without influencing the ability of the oligonucleotide tohybridise to the target region. In addition, variants may readily bedesigned which hybridise close to, for example to within 50 nucleotides,the region of the plant genome where the specific oligonucleotidesdefined herein hybridise. In particular, this includes polynucleotideswhich encode the same polypeptide or amino acid sequence but which varyin nucleotide sequence by redundancy of the genetic code. The terms“polynucleotide variant” and “variant” also include naturally occurringallelic variants.

Nucleic Acid Constructs

The present invention includes nucleic acid constructs comprising thepolynucleotides of the invention, and vectors and host cells containingthese, methods of their production and use, and uses thereof. Thepresent invention refers to elements which are operably connected orlinked. “Operably connected” or “operably linked” and the like refer toa linkage of polynucleotide elements in a functional relationship.Typically, operably connected nucleic acid sequences are contiguouslylinked and, where necessary to join two protein coding regions,contiguous and in reading frame. A coding sequence is “operablyconnected to” another coding sequence when RNA polymerase willtranscribe the two coding sequences into a single RNA, which iftranslated is then translated into a single polypeptide having aminoacids derived from both coding sequences. The coding sequences need notbe contiguous to one another so long as the expressed sequences areultimately processed to produce the desired protein.

As used herein, the term “cis-acting sequence”, “cis-acting element” or“cis-regulatory region” or “regulatory region” or similar term shall betaken to mean any sequence of nucleotides, which when positionedappropriately and connected relative to an expressible genetic sequence,is capable of regulating, at least in part, the expression of thegenetic sequence. Those skilled in the art will be aware that acis-regulatory region may be capable of activating, silencing,enhancing, repressing or otherwise altering the level of expressionand/or cell-type-specificity and/or developmental specificity of a genesequence at the transcriptional or post-transcriptional level. Inpreferred embodiments of the present invention, the cis-acting sequenceis an activator sequence that enhances or stimulates the expression ofan expressible genetic sequence.

“Operably connecting” a promoter or enhancer element to a transcribablepolynucleotide means placing the transcribable polynucleotide (e.g.,protein-encoding polynucleotide or other transcript) under theregulatory control of a promoter, which then controls the transcriptionof that polynucleotide. In the construction of heterologouspromoter/structural gene combinations, it is generally preferred toposition a promoter or variant thereof at a distance from thetranscription start site of the transcribable polynucleotide which isapproximately the same as the distance between that promoter and theprotein coding region it controls in its natural setting; i.e., the genefrom which the promoter is derived. As is known in the art, somevariation in this distance can be accommodated without loss of function.Similarly, the preferred positioning of a regulatory sequence element(e.g., an operator, enhancer etc) with respect to a transcribablepolynucleotide to be placed under its control is defined by thepositioning of the element in its natural setting; i.e., the genes fromwhich it is derived.

“Promoter” or “promoter sequence” as used herein refers to a region of agene, generally upstream (5′) of the RNA encoding region, which controlsthe initiation and level of transcription in the cell of interest. A“promoter” includes the transcriptional regulatory sequences of aclassical genomic gene, such as a TATA box and CCAAT box sequences, aswell as additional regulatory elements (i.e., upstream activatingsequences, enhancers and silencers) that alter gene expression inresponse to developmental and/or environmental stimuli, or in atissue-specific or cell-type-specific manner A promoter is usually, butnot necessarily (for example, some PolIII promoters), positionedupstream of a structural gene, the expression of which it regulates.Furthermore, the regulatory elements comprising a promoter are usuallypositioned within 2 kb of the start site of transcription of the gene.Promoters may contain additional specific regulatory elements, locatedmore distal to the start site to further enhance expression in a cell,and/or to alter the timing or inducibility of expression of a structuralgene to which it is operably connected.

“Constitutive promoter” refers to a promoter that directs expression ofan operably linked transcribed sequence in many or all tissues of anorganism such as a plant. The term constitutive as used herein does notnecessarily indicate that a gene is expressed at the same level in allcell types, but that the gene is expressed in a wide range of celltypes, although some variation in level is often detectable. “Selectiveexpression” as used herein refers to expression almost exclusively inspecific organs of, for example, the plant, such as, for example,endosperm, embryo, leaves, fruit, tubers or root. In a preferredembodiment, a promoter is expressed selectively or preferentially inleaves and/or stems of a plant, preferably a cereal plant. Selectiveexpression may therefore be contrasted with constitutive expression,which refers to expression in many or all tissues of a plant under mostor all of the conditions experienced by the plant.

Selective expression may also result in compartmentation of the productsof gene expression in specific plant tissues, organs or developmentalstages. Compartmentation in specific subcellular locations such as theplastid, cytosol, vacuole, or apoplastic space may be achieved by theinclusion in the structure of the gene product of appropriate signals,eg. a signal peptide, for transport to the required cellularcompartment, or in the case of the semi-autonomous organelles (plastidsand mitochondria) by integration of the transgene with appropriateregulatory sequences directly into the organelle genome.

A “tissue-specific promoter” or “organ-specific promoter” is a promoterthat is preferentially expressed in one tissue or organ relative to manyother tissues or organs, preferably most if not all other tissues ororgans in, for example, a plant. Typically, the promoter is expressed ata level 10-fold higher in the specific tissue or organ than in othertissues or organs.

In an embodiment, the promoter is a stem-specific promoter or a promoterwhich directs gene expression in an aerial part of the plant (greentissue specific promoter) such as a ribulose-1,5-bisphosphatecarboxylase oxygenase (RUBISCO) promoter.

Examples of stem-specific promoters include, but are not limited tothose described in U.S. Pat. No. 5,625,136, and Bam et al. (2008).

The promoters contemplated by the present invention may be native to thehost plant to be transformed or may be derived from an alternativesource, where the region is functional in the host plant. Other sourcesinclude the Agrobacterium T-DNA genes, such as the promoters of genesfor the biosynthesis of nopaline, octapine, mannopine, or other opinepromoters, tissue specific promoters (see, e.g., U.S. Pat. No. 5,459,252and WO 91/13992); promoters from viruses (including host specificviruses), or partially or wholly synthetic promoters. Numerous promotersthat are functional in mono- and dicotyledonous plants are well known inthe art (see, for example, Greve, 1983; Salomon et al., 1984; Garfinkelet al., 1983; Barker et al., 1983); including various promoters isolatedfrom plants and viruses such as the cauliflower mosaic virus promoter(CaMV 35S, 19S). Non-limiting methods for assessing promoter activityare disclosed by Medberry et al. (1992, 1993), Sambrook et al. (1989,supra) and U.S. Pat. No. 5,164,316.

Alternatively or additionally, the promoter may be an inducible promoteror a developmentally regulated promoter which is capable of drivingexpression of the introduced polynucleotide at an appropriatedevelopmental stage of the, for example, plant. Other cis-actingsequences which may be employed include transcriptional and/ortranslational enhancers. Enhancer regions are well known to personsskilled in the art, and can include an ATG translational initiationcodon and adjacent sequences. When included, the initiation codon shouldbe in phase with the reading frame of the coding sequence relating tothe foreign or exogenous polynucleotide to ensure translation of theentire sequence if it is to be translated. Translational initiationregions may be provided from the source of the transcriptionalinitiation region, or from a foreign or exogenous polynucleotide. Thesequence can also be derived from the source of the promoter selected todrive transcription, and can be specifically modified so as to increasetranslation of the mRNA.

The nucleic acid construct of the present invention may comprise a 3′non-translated sequence from about 50 to 1,000 nucleotide base pairswhich may include a transcription termination sequence. A 3′non-translated sequence may contain a transcription termination signalwhich may or may not include a polyadenylation signal and any otherregulatory signals capable of effecting mRNA processing. Apolyadenylation signal functions for addition of polyadenylic acidtracts to the 3′ end of a mRNA precursor. Polyadenylation signals arecommonly recognized by the presence of homology to the canonical form 5′AATAAA-3′ although variations are not uncommon. Transcriptiontermination sequences which do not include a polyadenylation signalinclude terminators for Poll or PolIII RNA polymerase which comprise arun of four or more thymidines. Examples of suitable 3′ non-translatedsequences are the 3′ transcribed non-translated regions containing apolyadenylation signal from an octopine synthase (ocs) gene or nopalinesynthase (nos) gene of Agrobacterium tumefaciens (Bevan et al., 1983).Suitable 3′ non-translated sequences may also be derived from plantgenes such as the ribulose-1,5-bisphosphate carboxylase (ssRUBISCO)gene, although other 3′ elements known to those of skill in the art canalso be employed.

As the DNA sequence inserted between the transcription initiation siteand the start of the coding sequence, i.e., the untranslated 5′ leadersequence (5′UTR), can influence gene expression if it is translated aswell as transcribed, one can also employ a particular leader sequence.Suitable leader sequences include those that comprise sequences selectedto direct optimum expression of the foreign or endogenous DNA sequence.For example, such leader sequences include a preferred consensussequence which can increase or maintain mRNA stability and preventinappropriate initiation of translation as for example described byJoshi (1987).

Vectors

The present invention includes use of vectors for manipulation ortransfer of genetic constructs. By “chimeric vector” is meant a nucleicacid molecule, preferably a DNA molecule derived, for example, from aplasmid, bacteriophage, or plant virus, into which a nucleic acidsequence may be inserted or cloned. A vector preferably isdouble-stranded DNA and contains one or more unique restriction sitesand may be capable of autonomous replication in a defined host cellincluding a target cell or tissue or a progenitor cell or tissuethereof, or capable of integration into the genome of the defined hostsuch that the cloned sequence is reproducible. Accordingly, the vectormay be an autonomously replicating vector, i.e., a vector that exists asan extrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g., a linear or closed circular plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into a cell,is integrated into the genome of the recipient cell and replicatedtogether with the chromosome(s) into which it has been integrated. Avector system may comprise a single vector or plasmid, two or morevectors or plasmids, which together contain the total DNA to beintroduced into the genome of the host cell, or a transposon. The choiceof the vector will typically depend on the compatibility of the vectorwith the cell into which the vector is to be introduced. The vector mayalso include a selection marker such as an antibiotic resistance gene, aherbicide resistance gene or other gene that can be used for selectionof suitable transformants Examples of such genes are well known to thoseof skill in the art.

The nucleic acid construct of the invention can be introduced into avector, such as a plasmid. Plasmid vectors typically include additionalnucleic acid sequences that provide for easy selection, amplification,and transformation of the expression cassette in prokaryotic andeukaryotic cells, e.g., pUC-derived vectors, pSK-derived vectors,pGEM-derived vectors, pSP-derived vectors, pBS-derived vectors, orbinary vectors containing one or more T-DNA regions. Additional nucleicacid sequences include origins of replication to provide for autonomousreplication of the vector, selectable marker genes, preferably encodingantibiotic or herbicide resistance, unique multiple cloning sitesproviding for multiple sites to insert nucleic acid sequences or genesencoded in the nucleic acid construct, and sequences that enhancetransformation of prokaryotic and eukaryotic (especially plant) cells.

By “marker gene” is meant a gene that imparts a distinct phenotype tocells expressing the marker gene and thus allows such transformed cellsto be distinguished from cells that do not have the marker. A selectablemarker gene confers a trait for which one can “select” based onresistance to a selective agent (e.g., a herbicide, antibiotic,radiation, heat, or other treatment damaging to untransformed cells). Ascreenable marker gene (or reporter gene) confers a trait that one canidentify through observation or testing, i.e., by “screening” (e.g.,β-glucuronidase, luciferase, GFP or other enzyme activity not present inuntransformed cells). The marker gene and the nucleotide sequence ofinterest do not have to be linked.

To facilitate identification of transformants, the nucleic acidconstruct desirably comprises a selectable or screenable marker gene as,or in addition to, the foreign or exogenous polynucleotide. The actualchoice of a marker is not crucial as long as it is functional (i.e.,selective) in combination with the plant cells of choice. The markergene and the foreign or exogenous polynucleotide of interest do not haveto be linked, since co-transformation of unlinked genes as, for example,described in U.S. Pat. No. 4,399,216 is also an efficient process inplant transformation.

Examples of bacterial selectable markers are markers that conferantibiotic resistance such as ampicillin, erythromycin, chloramphenicolor tetracycline resistance, preferably kanamycin resistance. Exemplaryselectable markers for selection of plant transformants include, but arenot limited to, a hyg gene which encodes hygromycin B resistance; aneomycin phosphotransferase (nptII) gene conferring resistance tokanamycin, paromomycin, G418; a glutathione-S-transferase gene from ratliver conferring resistance to glutathione derived herbicides as, forexample, described in EP 256223; a glutamine synthetase gene conferring,upon overexpression, resistance to glutamine synthetase inhibitors suchas phosphinothricin as, for example, described in WO 87/05327, anacetyltransferase gene from Streptomyces viridochromogenes conferringresistance to the selective agent phosphinothricin as, for example,described in EP 275957, a gene encoding a 5-enolshikimate-3-phosphatesynthase (EPSPS) conferring tolerance to N-phosphonomethylglycine as,for example, described by Hinchee et al. (1988), a bar gene conferringresistance against bialaphos as, for example, described in WO91/02071; anitrilase gene such as bxn from Klebsiella ozaenae which confersresistance to bromoxynil (Stalker et al., 1988); a dihydrofolatereductase (DHFR) gene conferring resistance to methotrexate (Thillet etal., 1988); a mutant acetolactate synthase gene (ALS), which confersresistance to imidazolinone, sulfonylurea or other ALS-inhibitingchemicals (EP 154,204); a mutated anthranilate synthase gene thatconfers resistance to 5-methyl tryptophan; or a dalapon dehalogenasegene that confers resistance to the herbicide.

Preferred screenable markers include, but are not limited to, a uidAgene encoding a β-glucuronidase (GUS) enzyme for which variouschromogenic substrates are known, a β-galactosidase gene encoding anenzyme for which chromogenic substrates are known, an aequorin gene(Prasher et al., 1985), which may be employed in calcium-sensitivebioluminescence detection; a green fluorescent protein gene (Niedz etal., 1995) or derivatives thereof; a luciferase (luc) gene (Ow et al.,1986), which allows for bioluminescence detection, and others known inthe art. By “reporter molecule” as used in the present specification ismeant a molecule that, by its chemical nature, provides an analyticallyidentifiable signal that facilitates determination of promoter activityby reference to protein product.

Preferably, the nucleic acid construct is stably incorporated into thegenome of, for example, the plant. Accordingly, the nucleic acidcomprises appropriate elements which allow the molecule to beincorporated into the genome, or the construct is placed in anappropriate vector which can be incorporated into a chromosome of aplant cell.

One embodiment of the present invention includes a recombinant vector,which includes at least one polynucleotide molecule of the presentinvention, inserted into any vector capable of delivering the nucleicacid molecule into a host cell. Such a vector contains heterologousnucleic acid sequences, that is nucleic acid sequences that are notnaturally found adjacent to nucleic acid molecules of the presentinvention and that preferably are derived from a species other than thespecies from which the nucleic acid molecule(s) are derived. The vectorcan be either RNA or DNA, either prokaryotic or eukaryotic, andtypically is a virus or a plasmid.

A number of vectors suitable for stable transfection of plant cells orfor the establishment of transgenic plants have been described in, e.g.,Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987;Weissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989; and Gelvin et al., Plant Molecular Biology Manual, KluwerAcademic Publishers, 1990. Typically, plant expression vectors include,for example, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences and a dominant selectablemarker. Such plant expression vectors also can contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

The level of a protein of the invention may be modulated by increasingthe level of expression of a nucleotide sequence that codes for theprotein in a plant cell, or decreasing the level of expression of a geneencoding the protein in the plant, leading to modified pathogenresistance. The level of expression of a gene may be modulated byaltering the copy number per cell, for example by introducing asynthetic genetic construct comprising the coding sequence and atranscriptional control element that is operably connected thereto andthat is functional in the cell. A plurality of transformants may beselected and screened for those with a favourable level and/orspecificity of transgene expression arising from influences ofendogenous sequences in the vicinity of the transgene integration site.A favourable level and pattern of transgene expression is one whichresults in a substantial modification of pathogen resistance or otherphenotype. Alternatively, a population of mutagenized seed or apopulation of plants from a breeding program may be screened forindividual lines with altered pathogen resistance or other phenotypeassociated with pathogen resistance.

Recombinant Cells

Another embodiment of the present invention includes a recombinant cellcomprising a host cell transformed with one or more recombinantmolecules of the present invention, or progeny cells thereof.Transformation of a nucleic acid molecule into a cell can beaccomplished by any method by which a nucleic acid molecule can beinserted into the cell. Transformation techniques include, but are notlimited to, transfection, electroporation, microinjection, lipofection,adsorption, and protoplast fusion. A recombinant cell may remainunicellular or may grow into a tissue, organ or a multicellularorganism. Transformed nucleic acid molecules of the present inventioncan remain extrachromosomal or can integrate into one or more siteswithin a chromosome of the transformed (i.e., recombinant) cell in sucha manner that their ability to be expressed is retained. Preferred hostcells are plant cells, more preferably cells of a cereal plant, morepreferably barley or wheat cells, and even more preferably a wheat cell.

Transgenic Plants

The term “plant” as used herein as a noun refers to whole plants andrefers to any member of the Kingdom Plantae, but as used as an adjectiverefers to any substance which is present in, obtained from, derivedfrom, or related to a plant, such as for example, plant organs (e.g.leaves, stems, roots, flowers), single cells (e.g. pollen), seeds, plantcells and the like. Plantlets and germinated seeds from which roots andshoots have emerged are also included within the meaning of “plant”. Theterm “plant parts” as used herein refers to one or more plant tissues ororgans which are obtained from a plant and which comprises genomic DNAof the plant. Plant parts include vegetative structures (for example,leaves, stems), roots, floral organs/structures, seed (including embryo,cotyledons, and seed coat), plant tissue (for example, vascular tissue,ground tissue, and the like), cells and progeny of the same. The term“plant cell” as used herein refers to a cell obtained from a plant or ina plant and includes protoplasts or other cells derived from plants,gamete-producing cells, and cells which regenerate into whole plants.Plant cells may be cells in culture. By “plant tissue” is meantdifferentiated tissue in a plant or obtained from a plant (“explant”) orundifferentiated tissue derived from immature or mature embryos, seeds,roots, shoots, fruits, tubers, pollen, tumor tissue, such as crowngalls, and various forms of aggregations of plant cells in culture, suchas calli. Exemplary plant tissues in or from seeds are cotyledon, embryoand embryo axis. The invention accordingly includes plants and plantparts and products comprising these.

As used herein, the term “seed” refers to “mature seed” of a plant,which is either ready for harvesting or has been harvested from theplant, such as is typically harvested commercially in the field, or as“developing seed” which occurs in a plant after fertilisation and priorto seed dormancy being established and before harvest.

A “transgenic plant” as used herein refers to a plant that contains anucleic acid construct not found in a wild-type plant of the samespecies, variety or cultivar. That is, transgenic plants (transformedplants) contain genetic material (a transgene) that they did not containprior to the transformation. The transgene may include genetic sequencesobtained from or derived from a plant cell, or another plant cell, or anon-plant source, or a synthetic sequence. Typically, the transgene hasbeen introduced into the plant by human manipulation such as, forexample, by transformation but any method can be used as one of skill inthe art recognizes. The genetic material is preferably stably integratedinto the genome of the plant. The introduced genetic material maycomprise sequences that naturally occur in the same species but in arearranged order or in a different arrangement of elements, for examplean antisense sequence. Plants containing such sequences are includedherein in “transgenic plants”.

A “non-transgenic plant” is one which has not been genetically modifiedby the introduction of genetic material by recombinant DNA techniques.In a preferred embodiment, the transgenic plants are homozygous for eachand every gene that has been introduced (transgene) so that theirprogeny do not segregate for the desired phenotype.

As used herein, the term “compared to an isogenic plant”, or similarphrases, refers to a plant which is isogenic relative to the transgenicplant but without the transgene of interest. Preferably, thecorresponding non-transgenic plant is of the same cultivar or variety asthe progenitor of the transgenic plant of interest, or a sibling plantline which lacks the construct, often termed a “segregant”, or a plantof the same cultivar or variety transformed with an “empty vector”construct, and may be a non-transgenic plant. “Wild type”, as usedherein, refers to a cell, tissue or plant that has not been modifiedaccording to the invention. Wild-type cells, tissue or plants may beused as controls to compare levels of expression of an exogenous nucleicacid or the extent and nature of trait modification with cells, tissueor plants modified as described herein.

Transgenic plants, as defined in the context of the present inventioninclude progeny of the plants which have been genetically modified usingrecombinant techniques, wherein the progeny comprise the transgene ofinterest. Such progeny may be obtained by self-fertilisation of theprimary transgenic plant or by crossing such plants with another plantof the same species. This would generally be to modulate the productionof at least one protein defined herein in the desired plant or plantorgan. Transgenic plant parts include all parts and cells of said plantscomprising the transgene such as, for example, cultured tissues, callusand protoplasts.

Plants contemplated for use in the practice of the present inventioninclude both monocotyledons and dicotyledons. Target plants include, butare not limited to, the following: cereals (for example, wheat, barley,rye, oats, rice, maize, sorghum and related crops); beet (sugar beet andfodder beet); pomes, stone fruit and soft fruit (apples, pears, plums,peaches, almonds, cherries, strawberries, raspberries andblack-berries); leguminous plants (beans, lentils, peas, soybeans); oilplants (rape or other Brassicas, mustard, poppy, olives, sunflowers,safflower, flax, coconut, castor oil plants, cocoa beans, groundnuts);cucumber plants (marrows, cucumbers, melons); fibre plants (cotton,flax, hemp, jute); citrus fruit (oranges, lemons, grapefruit,mandarins); vegetables (spinach, lettuce, asparagus, cabbages, carrots,onions, tomatoes, potatoes, paprika); lauraceae (avocados, cinnamon,camphor); or plants such as maize, tobacco, nuts, coffee, sugar cane,tea, vines, hops, turf, bananas and natural rubber plants, as well asornamentals (flowers, shrubs, broad-leaved trees and evergreens, such asconifers). Preferably, the plant is a cereal plant, more preferablywheat, rice, maize, triticale, oats, sorghum or barley, even morepreferably wheat.

As used herein, the term “wheat” refers to any species of the GenusTriticum, including progenitors thereof, as well as progeny thereofproduced by crosses with other species. Wheat includes “hexaploid wheat”which has genome organization of AABBDD, comprised of 42 chromosomes,and “tetraploid wheat” which has genome organization of AABB, comprisedof 28 chromosomes. Hexaploid wheat includes T. aestivum, T. spelta, T.macha, T. compactum, T. sphaerococcum, T. vavilovii, and interspeciescross thereof. A preferred species of hexaploid wheat is T. aestivum sspaestivum (also termed “breadwheat”). Tetraploid wheat includes T. durum(also referred to herein as durum wheat or Triticum turgidum ssp.durum), T. dicoccoides, T. dicoccum, T. polonicum, and interspeciescross thereof. In addition, the term “wheat” includes potentialprogenitors of hexaploid or tetraploid Triticum sp. such as T. uartu, T.monococcum or T. boeoticum for the A genome, Aegilops speltoides for theB genome, and T. tauschii (also known as Aegilops squarrosa or Aegilopstauschii) for the D genome. Particularly preferred progenitors are thoseof the A genome, even more preferably the A genome progenitor is T.monococcum. A wheat cultivar for use in the present invention may belongto, but is not limited to, any of the above-listed species. Alsoencompassed are plants that are produced by conventional techniquesusing Triticum sp. as a parent in a sexual cross with a non-Triticumspecies (such as rye [Secale cereale]), including but not limited toTriticale.

As used herein, the term “barley” refers to any species of the GenusHordeum, including progenitors thereof, as well as progeny thereofproduced by crosses with other species. It is preferred that the plantis of a Hordeum species which is commercially cultivated such as, forexample, a strain or cultivar or variety of Hordeum vulgare or suitablefor commercial production of grain.

Transgenic plants, as defined in the context of the present inventioninclude plants (as well as parts and cells of said plants) and theirprogeny which have been genetically modified using recombinanttechniques to cause production of at least one polypeptide of thepresent invention in the desired plant or plant organ. Transgenic plantscan be produced using techniques known in the art, such as thosegenerally described in A. Slater et al., Plant Biotechnology—The GeneticManipulation of Plants, Oxford University Press (2003), and P. Christouand H. Klee, Handbook of Plant Biotechnology, John Wiley and Sons(2004).

In a preferred embodiment, the transgenic plants are homozygous for eachand every gene that has been introduced (transgene) so that theirprogeny do not segregate for the desired phenotype. The transgenicplants may also be heterozygous for the introduced transgene(s), suchas, for example, in F1 progeny which have been grown from hybrid seed.Such plants may provide advantages such as hybrid vigour, well known inthe art.

As used herein, the “other genetic markers” may be any molecules whichare linked to a desired trait of a plant. Such markers are well known tothose skilled in the art and include molecular markers linked to genesdetermining traits such disease resistance, yield, plant morphology,grain quality, dormancy traits, grain colour, gibberellic acid contentin the seed, plant height, flour colour and the like. Examples of suchgenes are the stripe rust resistance genes Yr10 or Yr17, the nematoderesistance genes such as Cre1 and Cre3, alleles at glutenin loci thatdetermine dough strength such as Ax, Bx, Dx, Ay, By and Dy alleles, theRht genes that determine a semi-dwarf growth habit and therefore lodgingresistance.

Four general methods for direct delivery of a gene into cells have beendescribed: (1) chemical methods (Graham et al., 1973); (2) physicalmethods such as microinjection (Capecchi, 1980); electroporation (see,for example, WO 87/06614, U.S. Pat. Nos. 5,472,869, 5,384,253, WO92/09696 and WO 93/21335); and the gene gun (see, for example, U.S. Pat.Nos. 4,945,050 and 5,141,131); (3) viral vectors (Clapp, 1993; Lu etal., 1993; Eglitis et al., 1988); and (4) receptor-mediated mechanisms(Curiel et al., 1992; Wagner et al., 1992).

Acceleration methods that may be used include, for example,microprojectile bombardment and the like. One example of a method fordelivering transforming nucleic acid molecules to plant cells ismicroprojectile bombardment. This method has been reviewed by Yang etal., Particle Bombardment Technology for Gene Transfer, Oxford Press,Oxford, England (1994). Non-biological particles (microprojectiles) thatmay be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like. A particular advantage ofmicroprojectile bombardment, in addition to it being an effective meansof reproducibly transforming monocots, is that neither the isolation ofprotoplasts, nor the susceptibility of Agrobacterium infection arerequired. A particle delivery system suitable for use with the presentinvention is the helium acceleration PDS-1000/He gun is available fromBio-Rad Laboratories. For the bombardment, immature embryos or derivedtarget cells such as scutella or calli from immature embryos may bearranged on solid culture medium.

In another alternative embodiment, plastids can be stably transformed.Method disclosed for plastid transformation in higher plants includeparticle gun delivery of DNA containing a selectable marker andtargeting of the DNA to the plastid genome through homologousrecombination (U.S. Pat. Nos. 5,451,513, 5,545,818, 5,877,402,5,932,479, and WO 99/05265.

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art (see, for example, U.S. Pat. Nos. 5,177,010, 5,104,310,5,004,863, 5,159,135). Further, the integration of the T-DNA is arelatively precise process resulting in few rearrangements. The regionof DNA to be transferred is defined by the border sequences, andintervening DNA is usually inserted into the plant genome.

Agrobacterium transformation vectors are capable of replication in E.coli as well as Agrobacterium, allowing for convenient manipulations asdescribed (Klee et al., Plant DNA Infectious Agents, Hohn and Schell,(editors), Springer-Verlag, New York, (1985): 179-203). Moreover,technological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate construction of vectors capable of expressingvarious polypeptide coding genes. The vectors described have convenientmulti-linker regions flanked by a promoter and a polyadenylation sitefor direct expression of inserted polypeptide coding genes and aresuitable for present purposes. In addition, Agrobacterium containingboth armed and disarmed Ti genes can be used for the transformations. Inthose plant varieties where Agrobacterium-mediated transformation isefficient, it is the method of choice because of the facile and definednature of the gene transfer.

A transgenic plant formed using Agrobacterium transformation methodstypically contains a single genetic locus on one chromosome. Suchtransgenic plants can be referred to as being hemizygous for the addedgene. More preferred is a transgenic plant that is homozygous for theadded structural gene; i.e., a transgenic plant that contains two addedgenes, one gene at the same locus on each chromosome of a chromosomepair. A homozygous transgenic plant can be obtained by sexually mating(selfing) an independent segregant transgenic plant that contains asingle added gene, germinating some of the seed produced and analyzingthe resulting plants for the gene of interest.

It is also to be understood that two different transgenic plants canalso be mated to produce offspring that contain two independentlysegregating exogenous genes. Selfing of appropriate progeny can produceplants that are homozygous for both exogenous genes. Back-crossing to aparental plant and out-crossing with a non-transgenic plant are alsocontemplated, as is vegetative propagation. Descriptions of otherbreeding methods that are commonly used for different traits and cropscan be found in Fehr, Breeding Methods for Cultivar Development, J.Wilcox (editor) American Society of Agronomy, Madison Wis. (1987).

Transformation of plant protoplasts can be achieved using methods basedon calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments. Application ofthese systems to different plant varieties depends upon the ability toregenerate that particular plant strain from protoplasts. Illustrativemethods for the regeneration of cereals from protoplasts are described(Fujimura et al., 1985; Toriyama et al., 1986; Abdullah et al., 1986).

Other methods of cell transformation can also be used and include butare not limited to introduction of DNA into plants by direct DNAtransfer into pollen, by direct injection of DNA into reproductiveorgans of a plant, or by direct injection of DNA into the cells ofimmature embryos followed by the rehydration of desiccated embryos.

The regeneration, development, and cultivation of plants from singleplant protoplast transformants or from various transformed explants iswell known in the art (Weissbach et al., Methods for Plant MolecularBiology, Academic Press, San Diego, (1988)). This regeneration andgrowth process typically includes the steps of selection of transformedcells, culturing those individualized cells through the usual stages ofembryonic development through the rooted plantlet stage. Transgenicembryos and seeds are similarly regenerated. The resulting transgenicrooted shoots are thereafter planted in an appropriate plant growthmedium such as soil.

The development or regeneration of plants containing the foreign,exogenous gene is well known in the art. Preferably, the regeneratedplants are self-pollinated to provide homozygous transgenic plants.Otherwise, pollen obtained from the regenerated plants is crossed toseed-grown plants of agronomically important lines. Conversely, pollenfrom plants of these important lines is used to pollinate regeneratedplants. A transgenic plant of the present invention containing a desiredexogenous nucleic acid is cultivated using methods well known to oneskilled in the art.

Methods for transforming dicots, primarily by use of Agrobacteriumtumefaciens, and obtaining transgenic plants have been published forcotton (U.S. Pat. Nos. 5,004,863, 5,159,135, 5,518,908); soybean (U.S.Pat. Nos. 5,569,834, 5,416,011); Brassica (U.S. Pat. No. 5,463,174);peanut (Cheng et al., 1996); and pea (Grant et al., 1995).

Methods for transformation of cereal plants such as wheat and barley forintroducing genetic variation into the plant by introduction of anexogenous nucleic acid and for regeneration of plants from protoplastsor immature plant embryos are well known in the art, see for example, CA2,092,588, AU 61781/94, AU 667939, U.S. Pat. No. 6,100,447, WO97/048814, U.S. Pat. Nos. 5,589,617, 6,541,257, and other methods areset out in WO 99/14314. Preferably, transgenic wheat or barley plantsare produced by Agrobacterium tumefaciens mediated transformationprocedures. Vectors carrying the desired nucleic acid construct may beintroduced into regenerable wheat cells of tissue cultured plants orexplants, or suitable plant systems such as protoplasts. The regenerablewheat cells are preferably from the scutellum of immature embryos,mature embryos, callus derived from these, or the meristematic tissue.

To confirm the presence of the transgenes in transgenic cells andplants, a polymerase chain reaction (PCR) amplification or Southern blotanalysis can be performed using methods known to those skilled in theart. Expression products of the transgenes can be detected in any of avariety of ways, depending upon the nature of the product, and includeWestern blot and enzyme assay. One particularly useful way to quantitateprotein expression and to detect replication in different plant tissuesis to use a reporter gene, such as GUS. Once transgenic plants have beenobtained, they may be grown to produce plant tissues or parts having thedesired phenotype. The plant tissue or plant parts, may be harvested,and/or the seed collected. The seed may serve as a source for growingadditional plants with tissues or parts having the desiredcharacteristics.

Marker Assisted Selection

Marker assisted selection is a well recognised method of selecting forheterozygous plants required when backcrossing with a recurrent parentin a classical breeding program. The population of plants in eachbackcross generation will be heterozygous for the gene of interestnormally present in a 1:1 ratio in a backcross population, and themolecular marker can be used to distinguish the two alleles of the gene.By extracting DNA from, for example, young shoots and testing with aspecific marker for the introgressed desirable trait, early selection ofplants for further backcrossing is made whilst energy and resources areconcentrated on fewer plants. To further speed up the backcrossingprogram, the embryo from immature seeds (25 days post anthesis) may beexcised and grown up on nutrient media under sterile conditions, ratherthan allowing full seed maturity. This process, termed “embryo rescue”,used in combination with DNA extraction at the three leaf stage andanalysis of at least one Sr50 allele or variant that confers enhancedresistance to stem rust to the plant, allows rapid selection of plantscarrying the desired trait, which may be nurtured to maturity in thegreenhouse or field for subsequent further backcrossing to the recurrentparent.

Any molecular biological technique known in the art can be used in themethods of the present invention. Such methods include, but are notlimited to, the use of nucleic acid amplification, nucleic acidsequencing, nucleic acid hybridization with suitably labeled probes,single-strand conformational analysis (SSCA), denaturing gradient gelelectrophoresis (DGGE), heteroduplex analysis (HET), chemical cleavageanalysis (CCM), catalytic nucleic acid cleavage or a combination thereof(see, for example, Lemieux, 2000; Langridge et al., 2001). The inventionalso includes the use of molecular marker techniques to detectpolymorphisms linked to alleles of the (for example) Sr50 gene whichconfers enhanced resistance to stem rust. Such methods include thedetection or analysis of restriction fragment length polymorphisms(RFLP), RAPD, amplified fragment length polymorphisms (AFLP) andmicrosatellite (simple sequence repeat, SSR) polymorphisms. The closelylinked markers can be obtained readily by methods well known in the art,such as Bulked Segregant Analysis, as reviewed by Langridge et al.(2001).

In an embodiment, a linked loci for marker assisted selection is atleast within 1 cM, or 0.5 cM, or 0.1 cM, or 0.01 cM from a gene encodinga polypeptide of the invention.

The “polymerase chain reaction” (“PCR”) is a reaction in which replicatecopies are made of a target polynucleotide using a “pair of primers” or“set of primers” consisting of “upstream” and a “downstream” primer, anda catalyst of polymerization, such as a DNA polymerase, and typically athermally-stable polymerase enzyme. Methods for PCR are known in theart, and are taught, for example, in “PCR” (M. J. McPherson and S. GMoller (editors), BIOS Scientific Publishers Ltd, Oxford, (2000)). PCRcan be performed on cDNA obtained from reverse transcribing mRNAisolated from plant cells expressing a Sr50 gene or allele which confersenhanced resistance to stem rust. However, it will generally be easierif PCR is performed on genomic DNA isolated from a plant.

A primer is an oligonucleotide sequence that is capable of hybridisingin a sequence specific fashion to the target sequence and being extendedduring the PCR. Amplicons or PCR products or PCR fragments oramplification products are extension products that comprise the primerand the newly synthesized copies of the target sequences. Multiplex PCRsystems contain multiple sets of primers that result in simultaneousproduction of more than one amplicon. Primers may be perfectly matchedto the target sequence or they may contain internal mismatched basesthat can result in the introduction of restriction enzyme or catalyticnucleic acid recognition/cleavage sites in specific target sequences.Primers may also contain additional sequences and/or contain modified orlabelled nucleotides to facilitate capture or detection of amplicons.Repeated cycles of heat denaturation of the DNA, annealing of primers totheir complementary sequences and extension of the annealed primers withpolymerase result in exponential amplification of the target sequence.The terms target or target sequence or template refer to nucleic acidsequences which are amplified.

Methods for direct sequencing of nucleotide sequences are well known tothose skilled in the art and can be found for example in Ausubel et al.,(supra) and Sambrook et al., (supra). Sequencing can be carried out byany suitable method, for example, dideoxy sequencing, chemicalsequencing or variations thereof. Direct sequencing has the advantage ofdetermining variation in any base pair of a particular sequence.

Tilling

Plants of the invention can be produced using the process known asTILLING (Targeting Induced Local Lesions IN Genomes). In a first step,introduced mutations such as novel single base pair changes are inducedin a population of plants by treating seeds (or pollen) with a chemicalmutagen, and then advancing plants to a generation where mutations willbe stably inherited. DNA is extracted, and seeds are stored from allmembers of the population to create a resource that can be accessedrepeatedly over time.

For a TILLING assay, PCR primers are designed to specifically amplify asingle gene target of interest. Specificity is especially important if atarget is a member of a gene family or part of a polyploid genome. Next,dye-labeled primers can be used to amplify PCR products from pooled DNAof multiple individuals. These PCR products are denatured and reannealedto allow the formation of mismatched base pairs. Mismatches, orheteroduplexes, represent both naturally occurring single nucleotidepolymorphisms (SNPs) (i.e., several plants from the population arelikely to carry the same polymorphism) and induced SNPs (i.e., only rareindividual plants are likely to display the mutation). Afterheteroduplex formation, the use of an endonuclease, such as Cel I, thatrecognizes and cleaves mismatched DNA is the key to discovering novelSNPs within a TILLING population.

Using this approach, many thousands of plants can be screened toidentify any individual with a single base change as well as smallinsertions or deletions (1-30 bp) in any gene or specific region of thegenome. Genomic fragments being assayed can range in size anywhere from0.3 to 1.6 kb. At 8-fold pooling, 1.4 kb fragments (discounting the endsof fragments where SNP detection is problematic due to noise) and 96lanes per assay, this combination allows up to a million base pairs ofgenomic DNA to be screened per single assay, making TILLING ahigh-throughput technique.

TILLING is further described in Slade and Knauf (2005), and Henikoff etal. (2004). In addition to allowing efficient detection of mutations,high-throughput TILLING technology is ideal for the detection of naturalpolymorphisms. Therefore, interrogating an unknown homologous DNA byheteroduplexing to a known sequence reveals the number and position ofpolymorphic sites. Both nucleotide changes and small insertions anddeletions are identified, including at least some repeat numberpolymorphisms. This has been called Ecotilling (Comai et al., 2004).

Each SNP is recorded by its approximate position within a fewnucleotides. Thus, each haplotype can be archived based on its mobility.Sequence data can be obtained with a relatively small incremental effortusing aliquots of the same amplified DNA that is used for themismatch-cleavage assay. The left or right sequencing primer for asingle reaction is chosen by its proximity to the polymorphism.Sequencher software performs a multiple alignment and discovers the basechange, which in each case confirmed the gel band.

Ecotilling can be performed more cheaply than full sequencing, themethod currently used for most SNP discovery. Plates containing arrayedecotypic DNA can be screened rather than pools of DNA from mutagenizedplants. Because detection is on gels with nearly base pair resolutionand background patterns are uniform across lanes, bands that are ofidentical size can be matched, thus discovering and genotyping SNPs in asingle step. In this way, ultimate sequencing of the SNP is simple andefficient, made more so by the fact that the aliquots of the same PCRproducts used for screening can be subjected to DNA sequencing.

Plant/Grain Processing

Grain/seed of the invention, preferably cereal grain and more preferablywheat grain, or other plant parts of the invention, can be processed toproduce a food ingredient, food or non-food product using any techniqueknown in the art.

In one embodiment, the product is whole grain flour such as, forexample, an ultrafine-milled whole grain flour, or a flour made fromabout 100% of the grain. The whole grain flour includes a refined flourconstituent (refined flour or refined flour) and a coarse fraction (anultrafine-milled coarse fraction).

Refined flour may be flour which is prepared, for example, by grindingand bolting cleaned grain such as wheat or barley grain. The particlesize of refined flour is described as flour in which not less than 98%passes through a cloth having openings not larger than those of wovenwire cloth designated “212 micrometers (U.S. Wire 70)”. The coarsefraction includes at least one of: bran and germ. For instance, the germis an embryonic plant found within the grain kernel. The germ includeslipids, fiber, vitamins, protein, minerals and phytonutrients, such asflavonoids. The bran includes several cell layers and has a significantamount of lipids, fiber, vitamins, protein, minerals and phytonutrients,such as flavonoids. Further, the coarse fraction may include an aleuronelayer which also includes lipids, fiber, vitamins, protein, minerals andphytonutrients, such as flavonoids. The aleurone layer, whiletechnically considered part of the endosperm, exhibits many of the samecharacteristics as the bran and therefore is typically removed with thebran and germ during the milling process. The aleurone layer containsproteins, vitamins and phytonutrients, such as ferulic acid.

Further, the coarse fraction may be blended with the refined flourconstituent. The coarse fraction may be mixed with the refined flourconstituent to form the whole grain flour, thus providing a whole grainflour with increased nutritional value, fiber content, and antioxidantcapacity as compared to refined flour. For example, the coarse fractionor whole grain flour may be used in various amounts to replace refinedor whole grain flour in baked goods, snack products, and food products.The whole grain flour of the present invention (i.e.—ultrafine-milledwhole grain flour) may also be marketed directly to consumers for use intheir homemade baked products. In an exemplary embodiment, a granulationprofile of the whole grain flour is such that 98% of particles by weightof the whole grain flour are less than 212 micrometers.

In further embodiments, enzymes found within the bran and germ of thewhole grain flour and/or coarse fraction are inactivated in order tostabilize the whole grain flour and/or coarse fraction. Stabilization isa process that uses steam, heat, radiation, or other treatments toinactivate the enzymes found in the bran and germ layer. Flour that hasbeen stabilized retains its cooking characteristics and has a longershelf life.

In additional embodiments, the whole grain flour, the coarse fraction,or the refined flour may be a component (ingredient) of a food productand may be used to product a food product. For example, the food productmay be a bagel, a biscuit, a bread, a bun, a croissant, a dumpling, anEnglish muffin, a muffin, a pita bread, a quickbread, arefrigerated/frozen dough product, dough, baked beans, a burrito, chili,a taco, a tamale, a tortilla, a pot pie, a ready to eat cereal, a readyto eat meal, stuffing, a microwaveable meal, a brownie, a cake, acheesecake, a coffee cake, a cookie, a dessert, a pastry, a sweet roll,a candy bar, a pie crust, pie filling, baby food, a baking mix, abatter, a breading, a gravy mix, a meat extender, a meat substitute, aseasoning mix, a soup mix, a gravy, a roux, a salad dressing, a soup,sour cream, a noodle, a pasta, ramen noodles, chow mein noodles, lo meinnoodles, an ice cream inclusion, an ice cream bar, an ice cream cone, anice cream sandwich, a cracker, a crouton, a doughnut, an egg roll, anextruded snack, a fruit and grain bar, a microwaveable snack product, anutritional bar, a pancake, a par-baked bakery product, a pretzel, apudding, a granola-based product, a snack chip, a snack food, a snackmix, a waffle, a pizza crust, animal food or pet food.

In alternative embodiments, the whole grain flour, refined flour, orcoarse fraction may be a component of a nutritional supplement. Forinstance, the nutritional supplement may be a product that is added tothe diet containing one or more additional ingredients, typicallyincluding: vitamins, minerals, herbs, amino acids, enzymes,antioxidants, herbs, spices, probiotics, extracts, prebiotics and fiber.The whole grain flour, refined flour or coarse fraction of the presentinvention includes vitamins, minerals, amino acids, enzymes, and fiber.For instance, the coarse fraction contains a concentrated amount ofdietary fiber as well as other essential nutrients, such as B-vitamins,selenium, chromium, manganese, magnesium, and antioxidants, which areessential for a healthy diet. For example 22 grams of the coarsefraction of the present invention delivers 33% of an individual's dailyrecommend consumption of fiber. The nutritional supplement may includeany known nutritional ingredients that will aid in the overall health ofan individual, examples include but are not limited to vitamins,minerals, other fiber components, fatty acids, antioxidants, aminoacids, peptides, proteins, lutein, ribose, omega-3 fatty acids, and/orother nutritional ingredients. The supplement may be delivered in, butis not limited to the following forms: instant beverage mixes,ready-to-drink beverages, nutritional bars, wafers, cookies, crackers,gel shots, capsules, chews, chewable tablets, and pills. One embodimentdelivers the fiber supplement in the form of a flavored shake or malttype beverage, this embodiment may be particularly attractive as a fibersupplement for children.

In an additional embodiment, a milling process may be used to make amulti-grain flour or a multi-grain coarse fraction. For example, branand germ from one type of grain may be ground and blended with groundendosperm or whole grain cereal flour of another type of cereal.Alternatively bran and germ of one type of grain may be ground andblended with ground endosperm or whole grain flour of another type ofgrain. It is contemplated that the present invention encompasses mixingany combination of one or more of bran, germ, endosperm, and whole grainflour of one or more grains. This multi-grain approach may be used tomake custom flour and capitalize on the qualities and nutritionalcontents of multiple types of cereal grains to make one flour.

It is contemplated that the whole grain flour, coarse fraction and/orgrain products of the present invention may be produced by any millingprocess known in the art. An exemplary embodiment involves grindinggrain in a single stream without separating endosperm, bran, and germ ofthe grain into separate streams. Clean and tempered grain is conveyed toa first passage grinder, such as a hammermill, roller mill, pin mill,impact mill, disc mill, air attrition mill, gap mill, or the like. Aftergrinding, the grain is discharged and conveyed to a sifter. Further, itis contemplated that the whole grain flour, coarse fraction and/or grainproducts of the present invention may be modified or enhanced by way ofnumerous other processes such as: fermentation, instantizing, extrusion,encapsulation, toasting, roasting, or the like.

Malting

A malt-based beverage provided by the present invention involves alcoholbeverages (including distilled beverages) and non-alcohol beverages thatare produced by using malt as a part or whole of their startingmaterial. Examples include beer, happoshu (low-malt beer beverage),whisky, low-alcohol malt-based beverages (e.g., malt-based beveragescontaining less than 1% of alcohols), and non-alcohol beverages.

Malting is a process of controlled steeping and germination followed bydrying of the grain such as barley and wheat grain. This sequence ofevents is important for the synthesis of numerous enzymes that causegrain modification, a process that principally depolymerizes the deadendosperm cell walls and mobilizes the grain nutrients. In thesubsequent drying process, flavour and colour are produced due tochemical browning reactions. Although the primary use of malt is forbeverage production, it can also be utilized in other industrialprocesses, for example as an enzyme source in the baking industry, or asa flavouring and colouring agent in the food industry, for example asmalt or as a malt flour, or indirectly as a malt syrup, etc.

In one embodiment, the present invention relates to methods of producinga malt composition. The method preferably comprises the steps of:

(i) providing grain, such as barley or wheat grain, of the invention,

(ii) steeping said grain,

(iii) germinating the steeped grains under predetermined conditions and

(iv) drying said germinated grains.

For example, the malt may be produced by any of the methods described inHoseney (Principles of Cereal Science and Technology, Second Edition,1994: American Association of Cereal Chemists, St. Paul, Minn.) However,any other suitable method for producing malt may also be used with thepresent invention, such as methods for production of speciality malts,including, but limited to, methods of roasting the malt.

Malt is mainly used for brewing beer, but also for the production ofdistilled spirits. Brewing comprises wort production, main and secondaryfermentations and post-treatment. First the malt is milled, stirred intowater and heated. During this “mashing”, the enzymes activated in themalting degrade the starch of the kernel into fermentable sugars. Theproduced wort is clarified, yeast is added, the mixture is fermented anda post-treatment is performed.

EXAMPLES Example 1. Materials and Methods Plant Material and GrowthConditions

Plants of two wheat lines containing independent Sr50 introgressions,Gabo1BL.1RS and Gabo1DL.1RS-DR.A1, and of the mutants M2 (00.002), M7(00.007) and M13 (01.013) derived from these lines by y-irradiation andethyl methanesulfonate (EMS) treatment were used in the experimentsdescribed herein. These lines were as described by Rogowosky et al.(1991) and Mago et al. (2004). The Gabo1BL.1RS translocation wasbackcrossed five generations into wheat plants of the cultivarFederation to generate the resistance line Federation*5/Gabo1BL.1RS-1-1containing Sr50 in the absence of the Gabo background genes. Plants ofthe Gabo1BL.1RS line were resistant to North American and African Pgtrust isolates by the presence of the Sr50 resistance gene.

Nicotiana benthamiana plants were grown in a growth chamber at 22° C.with a 16 hours light period, 8 hours dark per 24 hours.

Marker Analysis and Rust Phenotyping

Markers developed from an Mla gene-containing BAC from chromosome 5H ofbarley (Wei et al., 1999) and used for mutant analysis herein were asdescribed by Mago et al. (2004). Stem rust phenotyping and mutantscreening were done on 1 week old seedlings with Puccinia graminis f. sptritici (Pgt) race 98-1 2,3,5,6 (Sydney University culture accession279) as described in Mago et al. (2009). To differentiate between Sr50and Sr31, plants were phenotyped with Pgt races TTKSK (Ug99), TTKST and98-1,2,3,5,7+50 (Sydney University culture accession 632). Transgenicplants were also phenotyped for leaf rust and stripe rust responses byinoculating seedlings with P. triticina pathotype 104-2,3,(6),(7),11(Sydney University culture accession 423) and P. striiformis f. sp.tritici pathotype 110 E143A+(Sydney University culture accession 444),respectively.

For rust infection assays to distinguish Sr50 from other resistancegenes with different specificities, one-week-old seedlings wereinoculated with Pgt races 34-2,4,5,7,11, (Plant Breeding Instituteaccession #760785); 34-2,12,13, (#840552); 126-5,6,7,11, (#334), TTKSK(04KEN156/04, Ug99), TTKST (06KEN19v3, Ug99+Sr24), QFCSC (03ND76C),TPMKC (74MN1409), TRTTF (06YEM34-1), TKKTP (13GER16-1), QCMJC(07WA140-515). Plants were also inoculated with P. triticina race104-2,3,(6),(7),11 (#890172) and P. striiformis f. sp. tritici race 110E143 A+(#861725). Infection types were scored as described (McIntosh,1995).

Generation of Genomic DNA Lambda Library

DraI-digested genomic DNA preparations from wheat plants of the lineGabo1DL.1RS were electrophoresed on a 1% agarose gel overnight and theregion of the gel containing fragments of between 9-13 kb was excisedand DNA extracted and purified from the gel. The resulting DNA wasligated to BamHI adaptors and cloned into an EMBL3 X-BamHI vector(Epicentre Technologies) and packaged using the MaxPlax (EpicentreTechnologies) lambda packaging extracts according to the manufacturer'sinstructions. The library was hybridised with a probe derived from theLRR-encoding region of Mla1 (B76: Mago et al., 2004) and positive cloneswere identified and sequenced.

BAC Screening and Sequence Analysis

A wheat-rye ditelosomic addition line comprising a rye chromosome 1RScarrying Sr50 in the background of wheat cv. Chinese Spring wasdescribed by Simkova et al. (2008). A BAC library was prepared from flowsorted chromosome 1RS from this ditelosomic addition line. This 1RSchromosome-specific library was screened by DNA hybridisation using theB76 probe as described below. Positive BAC clones were purified andfingerprinted using high-information content BAC fingerprintingaccording to standard methods. BAC DNA was prepared using a modifiedalkaline lysis protocol (Sinnett et al., 1998). BAC end sequencing ofclones in the minimal tiling path of the contig containing Sr50 wasperformed using primers designed to the pindigo BAC vector using Sangersequencing. BAC sequences were used to design specific PCR primers. FiveBAC clones were sequenced using the Roche 454 sequencing platform.Repeat sequences present in the assembled BACs were masked using theWheat Repeats Database(wheat.pw.usda.gov/ITMI/Repeats/blastrepeats3.html). Sequence reads wereassembled using Newbler v2.3. Non-repeated sequences were analysed forgenes using the gene prediction softwares FGENESH (www.softberry.com)and GENSCAN (genes.mit.edu/GENSCAN.html).

PCR Amplification of Sr50 Candidates

PCR amplification of candidate rust resistance genes from Gabo1DL.1RS-DR.A1 and various susceptible M2 mutants used primer pairsflanking the genes. Amplified sequences were compared for nucleotidevariations using multiple sequence alignment (CLUSTAL-EuropeanBioinformatics Institute—www.edi.ac.uk/Tools/sequence.html). RNAextraction, cDNA synthesis, 5′ and 3′ RACE (rapid amplification of cDNAends) were done using the methods described in Periyannan et al. (2013).Primers designed at the predicted 5′ and 3′ termini of Sr50 transcriptswere used for RT-PCR analysis. For 5′- and 3′-RACE, primers designed atthe 5′ and 3′ coding regions were used as the gene-specific primers.

Wheat Transformation

Two genomic DNA constructs each comprising Sr50 and therefore encodingthe Sr50 polypeptide were generated. The first contained a 7.5 kbfragment including 2.4 kbp upstream and 1.38 kbp downstream regionsrelative to the protein coding region of ScRGA1-A in the binary vectorpVecBarll. This fragment was amplified from Gabo1DL.1RS genomic DNAusing primers F1-R1 followed by nested primers F2-R2 listed in Table 2with PfuUltra II Fusion HS DNA Polymerase (Agilent Technologies) underthe manufacturer's recommended conditions. The second constructcontained a 9.8 kbp NotI fragment from BAC clone 180C18 including 4.2kbp upstream and 1.88kbp downstream regions relative to the proteincoding region and inserted into the binary vector pVecNeo.

TABLE 2 Primers used for PCR amplification ofSr50, ScRGA1 candidates and BAC ends. Forward Reverse Marker primerprimer Sr50-F1, R1 TAGCGCTGCT GATCCGCCGT CACATCCACC TGTCGGCATT TC TGT(SEQ ID (SEQ ID NO: 17) NO: 18) Sr50-F2, R2 ATTCATGCTT GGGCGTGACTTTATACTCAC GTGCTGCTT TAATATC (SEQ ID (SEQ ID NO: 20) NO: 19) Sr50-F3TTCAGTGAAG TTGCCGCTGT (SEQ ID NO: 21) SCRGA1-A-VIGS CGACAACTCCGACAAGGATC GGCAGATTTA GATAGTAATT GGTTC (SEQ ID (SEQ ID NO: 22) NO: 23)SCRGA1-A TCCACCTAAG GAGTTGGAAC RT-qPCR GTACCTTGAT CACCTTATA CTAC (SEQ ID(SEQ ID NO: 25) NO: 24) SCRGA1-A GCGCTGCCTG TAAAACAAAG RT-PCR GAATAAGGTCCCGCGGAAAA C (SEQ ID (SEQ ID NO: 26) NO: 27) RACE 5p, 3p GATTCCTGCCTCGGCATGAT TTTCTTAAAC GTCTTTGTTC AAGCCGA G (SEQ ID (SEQ ID NO: 28)NO: 29) p2D7-F-end GGCGGGCTGC GCCATCGGAT TAGTATTTCC CTGGAGAGAA (SEQ ID(SEQ ID NO: 30) NO: 31) p2D7-R-end CGTTGCAATG ACCGAGCTCG ATGTACCATATGTGCTCAA CG (SEQ ID (SEQ ID NO: 33) NO: 32) p2E7-F-end CAACAAGACGGTGCAGTTGC CACACCACCT AGAGGACCTG (SEQ ID (SEQ ID NO: 34) NO: 35)p2C2-F-end TTCGCAGGTT CTCCCGAATT CATCATGGTC GGAAAGTGGA (SEQ ID (SEQ IDNO: 36) NO: 37) p2C2-F-end CCTTGGCCTT TTGCCGGAAG TAGCTTGTGG CAAGAACTTT(SEQ ID (SEQ ID NO: 38) NO: 39) p1F7-F-end CGGAGTGTTT CCGATCCAGGGGATGAAAGG GGATATAGGT (SEQ ID (SEQ ID NO: 40) NO: 41) p1F7-R-endCTTCGTTAGG CATGCCTGAT AATGGCAGGT TCAATGTTGC (SEQ ID (SEQ ID NO: 42)NO: 43) p2B8-F-end GCACGCATGC GGGAAGCTCC ATGTAGTTGA TGGTTTGTTG (SEQ ID(SEQ ID NO: 44) NO: 45) p2B8-R-end ATCCGTGGGA AGATGGATTG GCTGTAGGTGGGCTGTGGAT (SEQ ID (SEQ ID NO: 46) NO: 47) p2C8-F-end CGCTCAGTTTATCGGAGTCG GCCGAAAAG TCGGAGAGAG (SEQ ID NO: 48) (SEQ ID NO: 49)p2C8-R-end GGTCCCTTGC TGTGATGGTG TCGTGAGTTC ATGCTTGTGC (SEQ ID (SEQ IDNO: 50) NO: 51) p2C7-F-end TCTGAAGCCG GGGAGTACTA GTCGAGTCTT GTCTCGCATC CA (SEQ ID (SEQ ID NO: 52) NO: 53) p2C7-R-end CATGGCTGCC TCACGCACGTACTCTCAAAG CAAGTCAAAA (SEQ ID (SEQ ID NO: 54) NO: 55) p2A3-F-endTGGTACTGTG GACGGCAAGA AAAGCGATTC TGGAGCAAGG TTATC A (SEQ ID (SEQ IDNO: 56) NO: 57) pIndigoBAC5 GGATGTGCTG CTCGTATGTT CAAGGCGATT GTGTGGAATTAAGTTGG GTGAGC (SEQ ID (SEQ ID NO: 58) NO: 59)

Binary vectors pVecNeo and pVecBarll are derivatives of pWBvec8 (Wang etal., 1998) in which the 35S promoter::hygromycin resistance gene wasreplaced with a 35S promoter::NPTII selectable marker gene derived frompCMneoSTL2 (Maas et al., 1997), or the bialaphos resistance gene (bar)coding region as a selection marker for plant transformation.

Transformation of the stem rust susceptible wheat cultivar Fielder wasdone using Agrobacterium tumefaciens strain GV3101 (pMP90) as described(Ishida et al., 2014; Richardson et al., 2014). TO transformants and T1progeny plants, including both plants which comprised the transgene andsegregants that lacked it as negative control plants, were tested forrust response with Pgt strain 98-1,2,3,5,6 as described above. Thepresence of the transgene and/or selectable marker gene was detected bySouthern blot hybridization as described (Mago et al., 2004). For this,a PCR amplified sequence from the 5′ end of Sr50 was used as agene-specific probe. Alternatively, transgene-specific PCR could havebeen carried out to detect the transgene.

Yeast Two-Hybrid Analysis

Yeast two-hybrid experiments were performed in Saccharomyces cerevisiaereporter strain Hf7c. The cDNAs encoding full length or truncated Sr50were cloned at EcoRI-XhoI sites of pGBKT7 and pGADT7 (Clontech). Yeasttransformation was performed according to Gietz and Woods (2002) withco-transformants selected on SD media lacking leucine and tryptophan.The interaction analysis was performed by plating the yeast cells onmedia lacking leucine, tryptophan and histidine and incubating theplates at 30° C. for 3-4 days.

Constructs for in Planta Expression

All PCR products used for cloning were generated using PhusionHigh-Fidelity DNA Polymerase (Finnzymes) with primers listed in Table 2.Molecular cloning was performed using Gateway recombination (LifeTechnologies) or Quickchange Site-Directed Mutagenesis (Stratagene). Forthe creation of Gateway entry clones, pDONR207 (Life Technologies) wasused. For Agrobacterium tumefaciens (agro) infiltration experiments,pBIN19-35S::GTW:3HA, pBIN19-35S::GTW:CFP (Cesari et al. 2014),pAM-PAT-35s::GTW:YFP:NLS; pAM-PAT-35s::GTW:YFP:nls;pAM-PAT-35s::GTW:YFPv: NES and pAM-PAT-35s::GTW:YFPv:nes were used.

A functional NES from HIV Rev (NES: LQLPPLERLTL; SEQ ID NO:15) andnon-functional nes (LQAPPAERATL; SEQ ID NO:16) (Wen et al., 1995) wereintroduced in the pAM-PAT-35s-GWY-YFPv vector (Bernoux et al., 2008) atthe C-terminus end of the YFPv. YFPv-NES/nes fragments werePCR-amplified using a forward primer containing a 3′ SmaI site and areverse primer containing a 5′ XbaI site as well as the NES or nessequence. Corresponding PCR products were ligated intopAM-PAT-35s-GWY-YFPv cut with SmaI/XbaI to replace the original YFPv byYFPv-NES/nes fusions.

Transient Protein Expression and Cell Death Assays in N. benthamiana

For Agrobacterium-mediated transformation of N. benthamiana leaf cells,cultures of Agrobacterium strain GV3101 transformed with the geneticconstruct pMP90 were grown in Luria-Bertani liquid medium containing 50mg ml-1 rifampicin, 15 mg ml-1 gentamycin and 25 mg ml-1 kanamycin at28° C. for 24 hours. The constructs providing for expression of NLS-,NES-, nls- and nes-fused proteins were transformed in A. tumefaciensstrain GV3103 and grown as described above with addition of 25 mg ml-1of carbenicillin. Bacteria were harvested by centrifugation, resuspendedin infiltration medium (10 mM MES pH 5.6, 10 mM MgCl2 and 150 μMacetosyringone) to an OD600 nm ranging from 0.5 to 1, and incubated for2 hours at room temperature before leaf infiltration. The infiltratedplants were incubated in growth chambers under controlled conditions forco-immunoprecipitation experiments and cell death assays. Fordocumentation of cell death, leaves were photographed 3-5 days afterinfiltration.

Confocal Microscopy

N. benthamiana epidermal cells were observed under a confocal microscope(TCS SP8; Leica) 20 hours after infiltration. Specific YFP fluorescencewas detected using the following spectral settings: excitation, 488 nm;detection, 515-545 nm. Auto-fluorescence of the chloroplasts wasdetected at 670-730 nm. All images were acquired using a water immersionlens (HC PL APO 63x/1.20 W CORR CS2, Leica).

Protein Extraction Western Blot and Co-Immunoprecipitation

Protein extraction from N. benthamiana leaves and co-immunoprecipitationexperiments were performed as described by Cesari et al. (2014). Forimmunoblot analysis, proteins were separated by SDS-PAGE and transferredto a nitrocellulose membrane (Pall). Membranes were blocked in 5%skimmed milk and probed with anti-HA or anti-Myc mouse monoclonalantibodies (Roche), followed by goat anti-mouse antibodies conjugatedwith horseradish peroxidase (Pierce). Labelling was detected using theSuperSignal West Pico or Femto chemiluminescence kits (Pierce).Membranes were stained with Ponceau S to confirm equal loading.

Example 2. The Sr50 Gene Encodes a Unique Resistance SpecificityEffective Against Ug99

To determine whether the fungal resistance gene Sr50 conferred the sameresistance specificity as, or a different resistance specificity to,Sr31, wheat plants of defined lines carrying one or other of thesegenes, or none of these genes as a control, were tested for theirreaction to a set of Pgt strains of different virulence specificities asdescribed in Example 1. Mutant plants carrying mutations in the Sr50gene were also tested; these were expected to be susceptible to Ptgstrains carrying the avirulence gene having Sr50 specificity. The datafrom the inoculation tests are summarised in Table 3.

TABLE 3 Infection types produced by lines containing Sr31 and Sr50 lineswhen inoculated with Pgt strain TTKSK (Ug99). Race of Pgt inoculum Plantname/genotype QFCS TPMK TTKSK TTKST Federation 3+ 4 4 4Federation*4/Kavkaz Sr31 1   1+ 4 4 Federation*4/Kavkaz -sr31 3+ 4 4 4(02.010-2) Gabo  1−;  2++ 2 2 Gabo 1DL.1RS- DR.A1 Sr50 ;1−  ;1  1 1 Gabo1BL.1RS Sr50 ;1   1 1 1 Gabo 1BL.1RS sr50 (M7) 0;   2++ 2 2

The results showed that while Sr31 was, as expected, ineffective inproviding resistance to Ptg strains Ug99 (TTKSK) and its derivativeTTKST, the Sr50 gene in the genetic background of the wheat variety Gaboprovided effective resistance to these strains, yielding an infectiontype 1 (Table 3). A mutant of Sr50 in this same genetic background (M7in Table 3) showed a similar phenotype (infection type 2) to the Gaboparent lacking the Sr50 gene, indicating that the mutation in the Sr50gene in the mutant inactivated the resistance gene. With someinoculations, an intermediate infection phenotype was observed in plantsof these lines—this was due to the presence of other stem rustresistance genes in the Gabo background that conferred partialresistance to Ug99 derivatives.

To aid in specifically testing for the Sr50 gene, a spontaneous mutantPgt strain with virulence for Sr50, designated 98-1,2,3,5,6+Sr50(available from Plant Breeding Institute accession #130176) was isolatedfrom a single pustule observed after infection of Sr50 plants withstrain 98-1,2,3,5,6 (#781219). Infection of a set of differential wheatlines, each containing different R genes, confirmed that the mutant Pgtstrain was identical to the parental isolate but with virulence to Sr50.This pair of Pgt strains could therefore be used to specifically detectthe presence of Sr50.

The genotypic and phenotypic difference between the Sr31 and Sr50resistance genes in plants was further confirmed by inoculation of theplants with the parental Ptg and the mutant Ptg derivative,98-1,2,3,5,6+Sr50. The parental Ptg strain 98-1,2,3,5,6 was virulent onplants of cultivar Gabo, this cultivar having no resistance genesagainst this Ptg strain, and avirulent on plants containing individuallySr31 or Sr50. In contrast, the mutant Ptg 98-1,2,3,5,6+Sr50 retainedavirulence to Sr31 but was virulent to Sr50 (Table 4). Furthermore, theparental strain and the mutant Ptg were both avirulent to plantscontaining Sr33, a wheat ortholog of Sr50 (see below), as well as to theSr^(Amigo) (1AL.1RS) present in plants of variety Amigo. Therefore, itwas concluded that the mutant Sr50 Ptg strain had lost activity of theSr50 avirulence gene (AvrSr50), and that the Sr50 resistance geneencoded a unique resistance specificity which could be distinguishedfrom the other 1RS located stem rust resistance genes. It was alsoconcluded that Sr50 could be distinguished from the other stem rustresistance genes by inoculation of the plants with different Ptg races.

TABLE 4 Comparative infection types produced by various lines wheninfected with Pgt race 98-1, 2 , 3, 5, 6 and its Sr50-mutant. Ptg strainPlant name/genotype 98-12357 98-12357 + Sr50 Westonia (Sr33)  2− 2  Gabo1DL.1RS-DR.A1 (Sr50) 12= 3+ Gabo 1BL.1RS (Sr50) 12= 3+ Mildress (Sr31)12= ;1   Amigo (1AL.1RS) derivative (Sr^(Amigo)) 2 − 2 2 − 2

Example 3. Physical Mapping of the Sr50 Locus

Mago et al. (2004) observed that a DNA hybridisation probe designatedB76, derived from the LRR coding region of the barley powdery mildewresistance gene Mla1, detected at least 17 hybridising fragments inDraI-restricted genomic DNA at the Sr50 locus of wheat and that severalSr50 mutants with small deletions had lost just two of these fragmentsof about 10 and 12 kbp. In an attempt to isolate the Sr50 gene, a lambdaphage genomic DNA library was prepared as described in Example 1 fromplants of the Sr50 line Gabo1BL.1RS and screened. The lambda librarycontaining DraI fragments that ranged in size from 9 to 13 kbp. Usingthe B76 hybridisation probe, six unique clones were isolated from thelibrary and selected for further analysis (FIG. 1 ).

Sequence analysis showed that all of these encoded Mla-related CC-NB-LRRsequences. Three contained various truncations or frameshifts andtherefore could not encode full length CC-NB-LRR proteins and two weresequences with some similarity to the LRR region of the B76 probe. Thesixth, clone 5 (FIG. 1 ), contained a mostly full length gene with nointernal interruptions of the reading frame but was truncated at a DraIsite in the LRR-encoding region and therefore was missing the 3′ end ofthe predicted gene.

To improve physical coverage of the deleted region including Sr50, probeB76 was then used to screen a 1RS chromosome-specific BAC libraryconstructed from a Sr50-containing wheat-rye ditelosomic addition line,as described in Example 1. This probe identified 175 BAC clones.High-information content BAC fingerprinting grouped these into 6non-overlapping contigs based on 2, 3, 11, 24, 71 and 72 clones.Eighteen BACs forming the minimum tiling path of these contigs werescreened by PCR using primers specific to the Mla homolog in lambdaclone 5. By this, BAC p2D7 (FIG. 2 ) was identified as containing thissequence. The PCR product was also amplified from four BAC clonesoverlapping p2D7, p2E7 p2B12, p2B9 and p1H3, confirming the presence ofthe candidate gene in this contig (FIG. 2 ).

To map the Sr50 gene and its surrounding locus onto the physical contig,BACs forming the minimum tiling path from the contig were end-sequencedand PCR markers developed from the BAC ends were used to screen the Sr50parent and the deletion mutant M2 (00.002). This analysis revealed thatthe deletion in mutant M2, and therefore the Sr50 gene, was whollycontained in the region spanned by 3 BAC clones, namely p2D7, p2A3 andp2C2, with the proximal and distal ends of p2D7 and p2C2 respectivelyretained (FIG. 2 ).

These three BAC clones spanning the deletion, as well as BAC p2E7, whichwas wholly contained in p2D7 and the adjacent BAC, p2C7, were sequenced.Annotation of the nucleotide sequences from this 250 kbp regionidentified six Mla-related NB-LRR protein coding regions (open readingframes) within the deleted region, and a single NB-LRR ORF in theadjacent region on BAC p2C7 (FIG. 2 ). These were designated as ScRGA1-Ato ScRGA1-G. The ORFs varied in length from 3.6 to 12.1 kb and each hadeither one or two predicted introns.

They encoded predicted polypeptides in the range of 944 to 974 aminoacids. A single copy of a chymotrypsin inhibitor (Ci) gene, a homolog ofwhich was also present at the Mla locus of barley, was also detectedwithin the deleted region. This was consistent with previous DNAhybridization analyses which identified 4 copies of a related Ci gene on1RS, only one of which was deleted in the interstitial deletion mutants(Mago et al., 2004). The amino acid sequence encoded by the lambda clone5 was 100% identical to the ScRGA1-A amino acid sequence, but the otherlambda clones identified as described above did not correspond to any ofthe other ScRGA1 genes in the BAC contig.

Example 4. Identification of the Sr50 Gene

In addition to the M2 deletion mutant described above, the inventors hadpreviously isolated several EMS-derived Sr50 gene mutants that retainedall of the B76-hybridising fragments and therefore possibly representedpoint mutations of Sr50 (Mago et al. 2004). Two mutant plants, M7(00.007) and M13 (01.013), were recovered and progeny plants produced. Across between M7 and M13 plants produced no resistant progeny indicatingthey carried a mutation in the same gene. The six ScRGA1 genesidentified within the M2 deletion as described above were amplified fromM7 and from M13 as well as from Gabo1DL.1RS DR.A1 and sequenced. Onegene candidate of the six, namely ScRGA1-A, contained a single base pairdeletion in M7 and a 23 bp deletion in M13. Both mutations resulted intranslational frame shifts which led to premature stop codons in the ORF(FIG. 3 ). All the other candidate genes were identical in sequence inthe wild-type plants and the mutants M7 and M13. The identification ofmultiple, independent mutations in the same coding region indicated thatthe ScRGA1-A coding region corresponded to the Sr50 gene. Thisconclusion was confirmed by transformation experiments (see below).

Example 5. Transformation of Wheat with Sr50 Genes

To confirm that ScRGA1-A conferred Sr50 resistance, two constructscontaining this gene were used to transform plants of the stemrust-susceptible wheat cultivar Fielder as described in Example 1. Thefirst construct, pVecBarSr50 (SEQ ID NO: 12), contained a 7.9 kbPCR-amplified genomic sequence of ScRGA1-A including the native promoterand terminator (2.4 and 1.4 kbp, respectively) within the T-DNA. Thesecond construct, pVecNeoSr50 (SEQ ID NO: 11), contained a 9.7 kb NotIgenomic fragment from BAC p2D7 with larger 5′ and 3′ regions, within theT-DNA. Both constructs therefore contained the genomic form of ScRGA1-Aincluding all of its introns as well as the native promoter, but linkedto heterologous sequences within a chimeric T-DNA construct. Theconstructs were used to transform wheat and confirmed transgenic plantsand their progeny tested as described in Example 1.

Independent T0 transgenic plants and T1 progeny plants containing theT-DNA from pVecBarSr50 were inoculated with Pgt strains. The transgenicplants exhibited responses to Pgt typical of Sr50-mediated resistancewhereas segregant plants that lacked the transgene were as susceptibleas untransformed parental plants. Likewise, TO wheat lines transformedwith pVecNeoSr50 showed resistance typical of Sr50. To confirm that theobserved resistance phenotype was race specific and not due to generalenhancement of defense pathways, T1 progeny of four stem rust resistanttransgenic plants (lines 2, 10b, 13 and 19d) were tested for theirreactions to leaf rust and stripe rust by inoculation with the strainsdescribed in Example 1.

All transgenic progeny plants were as susceptible to leaf rust andstripe rust as the non-transformed controls. Moreover, the T1 progenypopulations showed a 3:1 segregation ratio (resistant:susceptible) forreaction to stem rust, indicative of Mendelian inheritance ratios forinsertion of a single transgene in the nuclear genome. The combinedmutational and complementation data conclusively demonstrated that thegene designated ScRGA1-A was the Sr50 resistance gene.

The experiment also proved that the gene provided rust resistance whenused to transform a cereal plant. Thus, as the skilled person wouldappreciate, the same or similar constructs (for example with differentpromoter regions) can be used to produce other cereal plants (such asrice, barley, maize or sorghum) which express Sr50 resulting inresistance to one or more races Puccinia graminis.

Example 6. Structure of Sr50 and Comparison to Barley and WheatFunctional Mla Genes

The presence of two predicted introns in the protein coding region ofSr50 was confirmed by RT-PCR and comparison of the cDNA and genomicnucleotide sequences. Rapid amplification of cDNA ends (RACE) analysis,carried out as described by Jordan et al. (2011), identified 5′ and 3′UTRs of 629 and 238 bp, respectively, containing 3 additional introns(FIG. 3 ). Sizes of the exons and introns of Sr50 are shown in FIG. 3 .

The predicted Sr50 polypeptide was 77-78% identical in amino acidsequence to polypeptides in the barley MLA protein family, some of whichconfer resistance to powdery mildew, not rust resistance. Phylogeneticanalysis of cereal Mla homologs using their amino acid sequences placedthe Sr50 protein in a clade with other ScRGA1 polypeptides as well aswith the orthologous proteins TmMla1 and Sr33 from diploid wheatspecies, T. monococcum and Ae. tauschii (FIG. 4 ). The extent of aminoacid sequence identity amongst ScRGA1 members ranged between 78-89%whereas the Sr50 polypeptide showed 81% and 80% identity with the TmMla1and Sr33 amino acid sequences, respectively. The Lr1, Lr10 and Lr21wheat leaf rust resistance proteins were highly divergent from theMla/Sr33/Sr50 clade (FIG. 4 ), with sequence identities ranging from 19%to 33%, indicating a lack of relatedness with leaf rust resistancepolypeptides. The leaf rust polypeptides were vastly different to therust resistance polypeptides and could be easily distinguished. Thepolypeptide encoded by the only other cloned wheat stem rust resistancegene, Sr35 from T. monococcum, showed 48% amino acid identity with Sr50.

Example 7. Presence of Sr50 Gene and Variants in Cereals

To detect the presence of the Sr50 gene in rye, the origin species ofthe Sr50 gene in wheat, 114 geographically diverse rye accessions werescreened by PCR with primers flanking Sr50. The PCR amplification wasdone using PfuUltra II Fusion HS DNA Polymerase and oligonucleotideprimers F1-R1 followed by nested PCR with primers F3-R3. These reactionsamplified a 4.17 kb PCR product including the entire Sr50 gene.

Amplification of Sr50 sequences occurred from only 10 of the 114accessions, indicating the presence of a Sr50 gene or a close homolog inthose 10 accessions. For five of those accessions, the amplified genewas identical to Sr50, while three of the others contained smallsubstitutions and two were disrupted by a sequence inversion. All ofthese rye accessions except for Dwarf Petkus R1 showed resistance toseveral races of stem rust, including the Sr50-virulent mutant,indicating the presence of additional Sr genes, which may be on otherchromosomes. Despite the sequence similarity of the Sr50 gene in most ofthese accessions, they contained diverse haplotypes of this complexlocus, suggesting extensive recombination within this cluster in rye.Both of the Sr31 and stripe rust resistance Yr9 genes occur in the same1RS region, and may also belong to this gene family Therefore, thislocus appears to be a hotspot for evolution of fungal resistancespecificities in cereals, including in wheat, barley and rye.

Example 8. Functional Analysis of Sr50 Polypeptide The Coiled-CoilDomain of MLA10, Sr33 and Sr50 is Sufficient for Induction of Cell Death

Previous functional analysis of the MLA N-terminal region showed that a225 amino acid fragment including the CC and part of the NB domainscould self-associate in yeast, whereas a 160 amino acid fragmentcontaining the CC domain alone was autoactive in signalling cell deathin planta. A 120 amino acid fragment, which was a truncated CC domain,could dimerise in vitro (Maekawa et al., 2011; Bai et al., 2012).

To investigate whether the wheat and rye orthologs Sr33 and Sr50functioned similarly to MLA and to determine the minimal functionalregion in cell death signalling, various N-terminal fragments of theSr50 and Sr33 polypeptides were fused to either a C-terminal HA or CFPtag. These fusion polypeptides were transiently expressed in N.benthamiana leaf cells under the control of the 35S promoter asdescribed in Example 1. The constructs that expressed the full CCdomains of Sr33 and Sr50, namely amino acids 1-160 and 1-163,respectively, triggered a strong cell death response in the leaf cellsthat was visible 40 hours after agro-infiltration as a leaf tissuenecrosis of the infiltrated zones.

Similar to previous reports (Makeawa et al., 2011; Bai et al., 2012),the corresponding domain form barley MLA, MLA101-160, also induced astrong cell death response that was visible within 24 hours whenexpressed in N. benthamiana leaf cells. Another construct made toexpress a positive control protein fusion, the rice autoactive CC-NB-LRRRGA4 (Cesari et al., 2014), also produced a strong cell death response,while a mutant variant of RGA4 did not cause a response in N.benthamiana leaf cells. Expression of the CC domains together with aportion of the NB domains of Sr33 (1-225), Sr50 (1-228) and MLA10(1-225) also triggered strong cell death responses. However, thetruncated CC domains of MLA10 (1-120), Sr33 (1-120) and Sr50 (1-123)fused to the HA tag did not induce cell death in N. benthamiana. Westernblot analysis showed that all fusion proteins were properly expressed inthe leaf cells.

Taken together, these results showed that the full CC domains of MLA10,Sr33 and Sr50 were required and sufficient for induction of cell deathsignalling, and although the truncated CC domain of MLA10 dimerized insolution and its crystal structure had been resolved (Maekawa et al.,2011), it was not sufficient to trigger a cell death response. Thesedata demonstrated that the function of the CC domain in each polypeptidewas to trigger the induction of cell death, which is associated with theresistance response in wheat stem cells when the pathogen is present.

The Coiled-Coil Domains of Sr33, Sr50 and Lr21 Form Homo-Complexes

Although the truncated MLA10 CC5-120 fragment was able to self-associatein solution to form a dimer, only the MLA10 CC-NB1-225 domain had beenshown to self-interact when tested in a yeast two-hybrid assay (Maekawaet al., 2011). Self-interaction had not been demonstrated with theminimal active CC1-160 domain. Therefore, yeast-two-hybrid experimentswere carried out to test for interaction between the truncated CC, CCand CC-NB fragments of MLA10 and Sr50 Immunoblotting showed that all theprotein fragments were expressed in the yeast cells in the experiments.The results showed that the full CC domains of MLA10 and Sr50 wererequired for self association in yeast, whereas no interaction wasdetected for the truncated CC domains of these proteins.

To determine whether the active CC domains of MLA10, Sr33 and Sr50self-associated in planta, combinations of the HA and CFP-tagged MLA10,Sr33 and Sr50 domains were co-expressed in N. benthamiana leaf cells andco-immunoprecipitation assays were performed. The CC1-284 domain of theunrelated Lr21 resistance polypeptide, which was known to be autoactivein planta, was also included in the experiment to test its ability toself-interact. The RGA4 CC domain (amino acids 1-171) fused to CFP wasused as a control for binding specificity Immunoblotting using anti-GFPand anti-HA antibodies showed that all of the polypeptides wereexpressed as intended, except for the MLA10 fusions, which was likelydue to the more rapid cell death response induced by these polypeptidesand their consequent degradation in the cells Immunoprecipitation wasused to assay for association of the co-expressed polypeptidesImmunoprecipitation with anti-GFP antibodies resulted in enrichment ofall CFP-fused proteins and Sr331-160:HA, Sr501-163:HA and Lr211-284:HAspecifically co-precipitated with Sr331-160:CFP, Sr501-163:CFP andLr211-284:CFP, respectively, but not with RGA41-171:CFP. Taken together,these results showed that the CC domains of Sr33, Sr50 and Lr21 had thecapability to form specific homo-complexes in planta, i.e. the couldself-associate.

Cytoplasmic Localization of MLA10, Sr33 and Sr50 Coiled-Coil Domains isRequired for Cell Death Induction

The MLA10 full length polypeptide and its CC-NB1-225 domain have beenshown to trigger cell-death signalling when localised in the cytoplasmof N. benthamiana leaf cells (Bai et al., 2012). To determine whetherthe shorter MLA10 CC1-160 domain, thought to be needed for cell deathinduction, by itself could induce cell death when localised in thecytoplasm and to test this feature for the Sr33 and Sr50 CC domains,those domains were transiently expressed in N. benthamiana fused to YFPalong with a nuclear localisation signal (NLS), a mutated NLS (nls), anuclear export signal (NES) or a mutated NES (nes). Upon expression ofthe YFP:NLS fused CC domains, specific YFP fluorescence was detectedexclusively in the nuclei of N. benthamiana cells. In contrast, the CCdomains fused to YFP:NES were effectively excluded from the nuclei, withYFP fluorescence detected in the cytosol and surrounding the nucleionly, while the mutated nls and nes variants allowed detection of YFPfluorescence in both the cytosol and the nuclei as intended. Cell deathassays in N. benthamiana leaf cells revealed that forced nuclearlocalization of all CC domains reduced their cell-death activity. Otherfusions with YFP:NES, YFP:nes or YFP:nls did not affect the cell-deathinducing activity of MLA10 or Sr33 CC domains. In the case of the Sr50CC domain, the mutant nls also showed some reduction in cell deathactivity relative to the NES and nes fusions. Protein expression of allconstructs was verified by immunoblotting. The NES fusion constructsconsistently showed lower protein accumulation. Nevertheless, theseconstructs gave the strongest cell death (hypersensitive response, HR)phenotype, suggesting that cell death and protein degradation processeswere already activated at the time of sampling. These results indicatedthat cytosolic localization of those CC domains was required for celldeath induction.

Example 9. Discussion Both Powdery Mildew and Rust Resistances areEncoded by Mla Genes

The Mla gene clade of CC-NB-LRR genes, originally identified onchromosome 1HS in barley as a powdery mildew resistance gene also occursin wheat, where orthologs include the powdery mildew resistance geneTmMla1 (Jordon et al., 2011) and the stem rust resistance gene Sr33(Periyannan et al., 2013). The genes occur as a small gene family ofabout 5 members in wheat and barley. DNA gel blot analysis has shownthat this family has greatly expanded to over 20 members on chromosomeIRS in rye (Mago et al., 2004). The experiments described above have nowshown that the Sr50 stem rust resistance gene is a member of the rye Mlaorthologous gene family

Although the wheat stem rust resistance genes Sr33 and Sr35 wereisolated previously (Periyannan et al., 2013; Saintenac et al., 2013),originating from wheat, the Sr50 gene was the first stem rust resistancegene to be cloned which originated from rye and which is effectiveagainst all known field races of Pgt including Ug99. High resolutionmapping and mutation studies have also placed both Sr31 and the striperust resistance gene Yr9 from Petkus rye IRS in the region homoeologousto Sr50 (Mago et al., 2005), suggesting that these genes may also belongto the Mla gene family Therefore, this multi-species locus appears to bea hotspot for evolution of resistance specificities in the cereals.

Subcellular Localization of NB-LRR Proteins and Cell Death Activation

Increasing knowledge about localization of NB-LRR proteins has revealedthat they can be observed and activated in a wide diversity of cellularcompartments. Some are exclusively nuclear such as RRS1 (Deslandes etal., 2003; Tasset et al., 2010), whereas others like RGA4 and RGA5 aremainly localized in the cytosol (Cesari et al., 2014). Rpm1 (Gao et al.,2011), RPS2 (Axtell and Staskawicz, 2003) and RPS5 (Qi et al., 2012) arelocated at the plasma membrane, and the flax L6 and M proteins occur onthe Golgi and tonoplast membranes, respectively (Takemoto et al., 2012).However, many show a nucleo-cytoplasmic localization such as N(Burch-Smith et al., 2007; Caplan et al., 2008), RPS4 (Wirthmueller etal., 2007), SNC1 (Cheng et al., 2009), Pik1/Pik2 (Zhai et al., 2014), Rx(Slootweg et al., 2010) and MLA10 (Shen et al., 2007).

In the case of the potato CC-NB-LRR polypeptide Rx that triggersrecognition of the Potato Virus X (PVX) coat protein (CP), it was shownthat CP activates Rx in the cytoplasm, and forced nuclear localizationof Rx severely compromised both virus resistance and cell death(Slootweg et al., 2010; Tameling et al., 2010). However, Rx nuclearexclusion only moderately reduced PVX resistance (Slootweg et al.,2010), suggesting that the cytosolic pool was most important, althoughboth nuclear and cytoplasmic pools of the resistance protein may havecontributed to resistance signalling. Conversely, another study on MLA10demonstrated that a nuclear localised receptor was sufficient to conferresistance to B. graminis in a transient single cell assay in barley,while a nuclear-excluded MLA10 was not (Shen et al., 2007). However,forced nuclear localisation of autoactive MLA protein variants impairedinduction of cell death whereas nuclear excluded variants weresufficient to trigger resistance (Bai et al., 2012). To explain this, ithas been proposed that MLA10-triggered cell death signalling and diseaseresistance signalling occur in different compartments (Bai et al.,2012). Alternatively, AVR_(A10) recognition may occur exclusively in thenucleus, explaining the requirement for a nuclear localised receptor toconfer resistance, with subsequent signalling events perhaps occurringin the cytoplasm.

Sr33 and, as demonstrated by the experiments described above, Sr50 arehomologs of MLA10 and using their minimal CC signaling domains, asimilar pattern of cell death induction was observed relative to thattriggered by the full length MLA10 protein or the MLA10 CC-NB domain(Bai et al., 2012). Therefore, it appeared that the responses triggeredby MLA10 in barley and its close wheat homologs were similar, suggestinga conserved cell death pathway triggered by different pathogens in twodifferent cereal species.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

This application claims priority from AU 2015904976 filed 1 Dec. 2015,the entire contents of which are incorporated herein by reference.

All publications discussed and/or referenced herein are incorporatedherein in their entirety.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

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1.-54. (canceled)
 55. A chimeric vector or nucleic acid constructcomprising a polynucleotide encoding a polypeptide which comprises aminoacids having a sequence as provided in SEQ ID NO:1, an amino acidsequence which is at least 90% identical to SEQ ID NO:1 or abiologically active fragment thereof, wherein the polypeptide confersresistance to one or more races of Puccinia graminis, wherein thepolynucleotide is operably linked to a promoter.
 56. The chimeric vectoror nucleic acid construct of claim 55, wherein the polypeptide comprisesamino acids having a sequence as provided in SEQ ID NO:1, an amino acidsequence which is at least 95% identical to SEQ ID NO:1 or abiologically active fragment thereof.
 57. The chimeric vector or nucleicacid construct of claim 55, wherein the polypeptide comprises aminoacids having a sequence as provided in SEQ ID NO:1, an amino acidsequence which is at least 97% identical to SEQ ID NO:1 or abiologically active fragment thereof.
 58. The chimeric vector or nucleicacid construct of claim 55, wherein the polypeptide comprises aminoacids having a sequence as provided in SEQ ID NO:1, an amino acidsequence which is at least 99% identical to SEQ ID NO:1 or abiologically active fragment thereof.
 59. The chimeric vector or nucleicacid construct of claim 55 which comprises one or more further exogenouspolynucleotides encoding another plant pathogen resistance polypeptide.60. The chimeric vector or nucleic acid construct of claim 55, whereinthe polypeptide comprises one, more or all of a coiled coil (CC) domain,an nucleotide binding (NB) domain and a leucine rich repeat (LRR)domain.
 61. The chimeric vector or nucleic acid construct of claim 55,wherein the biologically fragment is the leucine rich repeat (LRR)domain or the coiled coil (CC) domain of the polypeptide having asequence as provided in SEQ ID NO:1, or an amino acid sequence which isat least 90% identical to SEQ ID NO:1.
 62. A chimeric vector or nucleicacid construct comprising a polynucleotide encoding a polypeptide havingan LRR domain which comprises 5 to 20 repeats of the sequence xxLxLxxxx(SEQ ID NO:8) from amino acids having a sequence as provided in SEQ IDNO:1, wherein the polynucleotide is operably linked to a promoter.
 63. Amethod of producing a plant, the method comprising the steps of i)introducing the chimeric vector or nucleic acid construct of claim 55into a cell of a plant, ii) regenerating a plant from the cell, therebyproducing the plant.
 64. The method of claim 63 which further comprisesharvesting seed from the plant.
 65. The method of claim 63 which furthercomprises producing one or more progeny plants from the plant.
 66. Themethod of claim 63, wherein plant is a cereal plant.
 67. The method ofclaim 63, wherein plant is a wheat plant.
 68. A method of producing aplant, the method comprising the steps of i) crossing two parentalplants, wherein at least one plant comprises the chimeric vector ornucleic acid construct of claim 55, ii) screening one or more progenyplants from the cross for the presence or absence of the chimeric vectoror nucleic acid construct, and iii) selecting a progeny plant whichcomprise the chimeric vector or nucleic acid construct, therebyproducing the plant.
 69. A method for identifying a plant comprising thechimeric vector or nucleic acid construct of claim 55, the methodcomprising the steps of i) obtaining a nucleic acid sample from a plant,and ii) screening the sample for the presence or absence of the chimericvector or nucleic acid construct, wherein presence of the chimericvector or nucleic acid construct indicates that the plant is resistantto Puccinia graminis.
 70. A method of producing a plant part, the methodcomprising, a) growing a plant comprising a chimeric vector or nucleicacid construct of claim 55, and b) harvesting the plant part.
 71. Amethod of producing flour, wholemeal, starch or other product obtainedfrom seed, the method comprising; a) obtaining seed comprising thechimeric vector or nucleic acid construct of claim 55, and b) extractingthe flour, wholemeal, starch or other product.
 72. A method of preparinga food product, the method comprising mixing seed comprising thechimeric vector or nucleic acid construct of claim 55, or flour,wholemeal or starch from the seed, with another food ingredient.
 73. Useof a plant comprising the chimeric vector or nucleic acid construct ofclaim 55, or part thereof comprising the chimeric vector or nucleic acidconstruct, as animal feed, or to produce feed for animal consumption orfood for human consumption.
 74. Flour or wholemeal comprising thechimeric vector or nucleic acid construct of claim 55.